Automated nucleic acid assembly and introduction of nucleic acids into cells

ABSTRACT

In an illustrative embodiment, automated instruments comprising one or more flow-through electroporation devices or modules are provided to automate transformation of nucleic acids in live cells.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/548,208, entitled “Automated Nucleic Acid Assembly and Introductionof Nucleic Acids into Cells, filed Aug. 22, 2019, which is acontinuation of U.S. patent application Ser. No. 16/147,871, now U.S.Pat. No. 10,415,058 issued Sep. 17, 2019, entitled “Automated NucleicAcid Assembly and Introduction of Nucleic Acids into Cells, filed Sep.30, 2018, which claims priority to U.S. Patent Application Ser. No.62/566,374, entitled “Electroporation Device,” filed Sep. 30, 2017; U.S.Patent Application Ser. No. 62/566,375, entitled “ElectroporationDevice,” filed Sep. 30, 2017; U.S. Patent Application Ser. No.62/566,688, entitled “Introduction of Nucleic acids into Cells,” filedOct. 2, 2017; U.S. Patent Application Ser. No. 62/567,697, entitled“Automated Nucleic Acid Assembly and Introduction of Nucleic Acids intoCells,” filed Oct. 3, 2017; U.S. Patent Application Ser. No. 62/620,370,entitled “Automated Filtration and Manipulation of Viable Cells,” filedJan. 22, 2018; U.S. Patent Application Ser. No. 62/649,731, entitled“Automated Control of Cell Growth Rates for Induction andTransformation,” filed Mar. 29, 2018; U.S. Patent Application Ser. No.62/671,385, entitled “Automated Control of Cell Growth Rates forInduction and Transformation,” filed May 14, 2018; U.S. PatentApplication Ser. No. 62/648,130, entitled “Genomic Editing in AutomatedSystems,” filed Mar. 26, 2018; U.S. Patent Application Ser. No.62/657,651, entitled “Combination Reagent Cartridge and ElectroporationDevice,” filed Apr. 13, 2018; U.S. Patent Application Ser. No.62/657,654, entitled “Automated Cell Processing Systems ComprisingCartridges,” filed Apr. 13, 2018; and U.S. Patent Application Ser. No.62/689,068, entitled “Nucleic Acid Purification Protocol for Use inAutomated Cell Processing Systems,” filed Jun. 23, 2018. All aboveidentified applications are hereby incorporated by reference in theirentireties for all purposes.

BACKGROUND

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

A cell membrane constitutes the primary barrier for the transport ofmolecules and ions between the interior and the exterior of a cell.Electroporation, also known as electropermeabilization, substantiallyincreases the membrane permeability in the presence of a pulsed electricfield. The technique is more reproducible, universally applicable, andefficient than other physical methods and alternative biological andchemical techniques.

Conventional electroporation is typically conducted by exerting shortelectric pulses of defined intensity and duration to a cuvette equippedwith embedded electrodes inside. Potter H., Anal. Biochem., 1988, 174,361-373 The electrodes are commonly fabricated out of aluminum (Al),stainless-steel, platinum (Pt) or graphite, and arranged in aplate-to-plate manner. A pulse generator such as special capacitordischarge equipment is required to generate the high voltage pulses. Bytuning the electric parameters, electroporation efficiency and cellviability (for delivery) can be optimized. Canatella P J et al.,Biophys. J., 2001, 80, 755-764.

Although the traditional electroporation systems have been widely used,they require a high voltage input and suffer from adverse environmentalconditions such as electric field distortion, local pH variation, metalion dissolution and excess heat generation, resulting in lowelectroporation efficiency and/or cell viability.

In addition, the materials such as nucleic acids that are transformedinto cells need to exhibit the appropriate activity followingtransformation. This often requires the assembly of the materials to betransformed into a form that allows, e.g., recovery, expression,transcription, translation, etc. of the RNA and/or proteins encoded bythe nucleic acid. There is thus a need for automated methods ofintroducing assembled nucleic acids into cells in an automated fashion.The present invention addresses this need.

SUMMARY OF ILLUSTRATIVE EMBODIMENTS

The present disclosure is based on the development of automatedinstruments and systems for carrying out automated methods oftransformation of nucleic acids into cells. These methods can be used togenerate libraries of living cells of interest having the nucleic acidsintroduced therein. The novel, automated methods carried out using theinstruments and system of the disclosure can be used with a variety ofmaterials and techniques, and can be used with or without use of one ormore selectable markers. Optionally, the automated instrument alsocomprises a module for the automated assembly of the nucleic acids to betransformed into the cells.

In some aspects, the disclosure provides an instrument for automatedlive cell electroporation, the instrument having a receptacle configuredto receive nucleic acids to be delivered to the cells; a receptacleconfigured to receive live cells; an electroporation device forintroduction of the assembled nucleic acids into the cells, and aprocessor-based system configured to operate the instrument based onuser input. The automated introduction of the nucleic acids into thecells is preferably performed using one or more flow throughelectroporation (FTEP) devices, as described in more detail herein.

In some aspects, the disclosure provides an instrument for nucleic acidassembly and automated live cell electroporation, the instrument havinga receptacle configured to receive nucleic acids to be assembled anddelivered to the cells; an assembly module for the assembly of nucleicacids to be transformed into the cells; a receptacle configured toreceive live cells; an electroporation device for introduction of theassembled nucleic acids into the cells, and a processor-based systemconfigured to operate the instrument based on user input.

In specific embodiments, the instrument comprises two or moreflow-through electroporation devices that can introduce differentnucleic acids into populations of different cells in a single operationof the instrument.

In certain aspects, the instrument further provides a purificationmodule into which the assembled nucleic acids are transferred prior totransformation. The purification can remove unwanted components of thenucleic acid assembly mixture (e.g., salts, minerals) and optionallyconcentrate the assembled nucleic acids.

In specific aspects, the disclosure provides an instrument for automatedlive cell electroporation, the instrument having a receptacle configuredto receive live cells and nucleic acids to be delivered to the cells, anelectroporation device for introduction of the nucleic acids into thecells, and a processor-based system configured to operate the instrumentbased on user input.

In other specific aspects, the disclosure provides an instrument forautomated live cell electroporation, the instrument having a receptacleconfigured to receive live cells, a receptacle configured to receivenucleic acids to be delivered to the cells, an electroporation devicefor introduction of the nucleic acids into the cells, and aprocessor-based system configured to operate the instrument based onuser input.

In some aspects, the instruments of the disclosure further comprise acell growth unit. In other aspects, the instruments of the disclosurefurther comprise a cell selection unit. In yet other aspects, theinstruments of the disclosure further comprise a cell concentrationunit. In still other aspects, the instruments of the disclosure furthercomprise both a cell growth and a cell concentration unit. In specificaspects, the instruments of the disclosure further comprise a cellgrowth unit, a cell selection module and a cell concentration unit. Eachof the instruments may also optionally contain a cell wash function andan optional storage module for storage of the cells followingtransformation.

Accordingly, in one specific embodiment, the disclosure provides aninstrument for automated live cell electroporation, comprising: areceptacle configured to receive nucleic acids to be assembled anddelivered to the cells, an assembly module for the assembly of nucleicacids to be transformed into the cells; a purification module to removeunwanted components of the nucleic acid assembly; a receptacleconfigured to receive live cells; an electroporation device forintroduction of the assembled nucleic acids into the cells, and aprocessor-based system configured to operate the instrument based onuser input.

In other specific aspects, the disclosure provides an instrument forautomated live cell electroporation, comprising a receptacle configuredto receive live cells; a receptacle configured to receive nucleic acids;an assembly module for assembly of the nucleic acids prior totransformation; an electroporation device for introduction of thenucleic acids into the cells; and a processor-based system configured tooperate the instrument based on user input.

In some aspects, the instruments of the disclosure further comprise acell growth unit. In other aspects, the instruments of the disclosurefurther comprise a cell selection unit. In yet other aspects, theinstruments of the disclosure further comprise a cell concentrationunit. In still other aspects, the instruments of the disclosure furthercomprise both a cell growth and a cell concentration unit. In specificaspects, the instruments of the disclosure further comprise a cellgrowth unit, a cell selection module and a cell concentration unit. Eachof the instruments may also optionally contain a cell wash function andan optional storage module for storage of the cells followingtransformation. a combination reagent cartridge and electroporationdevice configured for use in an automated multi-module cell processingenvironment. The reagent cartridges include an electroporation device,as well as sample receptacles, reagent receptacles, waste receptaclesand the like, and a script for controlling a processor to dispensesamples and reagents contained in the receptacles, and to porate cellsin the electroporation device. Also described are kits including thecartridges, automated instruments including the reagent cartridges andmethods of using the reagent cartridges.

Thus, presented herein is an exemplary embodiment of an automatedinstrument comprising a reagent cartridge comprising a plurality ofreagent reservoirs, a flow-through electroporation device, and a scriptreadable by a processor for dispensing reagents located in the pluralityof reagent reservoirs and controlling the electroporation device,wherein the script comprises commands for retrieving reagents in thereagent cartridge and commands for electroporating cells in theflow-through electroporation device.

In some aspects of this embodiment, the flow-through electroporationdevice comprises an inlet and inlet channel for introduction of a cellsample to the flow-through electroporation (FTEP) device; an outlet andoutlet channel for exit of the cell sample from the FTEP device; aconstricted flow channel intersecting and positioned between the inletchannel and outlet channel; and two or more electrodes, wherein the twoor more electrodes are (a) positioned in the flow channel between theintersection of the flow channel with the first inlet channel and theintersection of the flow channel with the outlet channel and on eitherside of the constriction in the flow channel, (b) in fluid communicationwith the cell sample in the flow channel but are not in the flow path ofthe cell sample in the flow channel, and (c) configured to apply anelectric pulse or electric pulses to a cell sample.

Also in some aspects the script readable by a processor comprisescommands for performing one or more additional processes in theautomated instrument, and in some aspects, the script readable by aprocessor comprises commands for performing all processes in theautomated instrument.

In some aspects of this embodiment, the automated instrument furthercomprises a cell growth module, and in some aspects, the cell growthmodule comprises a rotating growth vial.

In some aspects the automated instrument further comprises a filtrationmodule, and in some aspects, the filtration module comprises a hollowfiber filter.

The automated instrument may also further comprise a recovery module,and in some aspects the recovery module comprises a cell growth modulecomprising a rotating growth vial.

In some aspects, the automated instrument further comprises a storagemodule, and/or a nucleic acid assembly module, where in some aspects thenucleic acid assembly module is a Gibson assembly module or a Gap Repairmodule.

In some aspects the automated instrument further comprises a processorto read the script.

Another embodiment of an automated instrument presented herein comprisesa reagent cartridge comprising a plurality of reagent reservoirs, aflow-through electroporation device, wherein the flow-throughelectroporation device comprises an inlet and inlet channel forintroduction of a cell sample to the flow-through electroporationdevice; an outlet and outlet channel for exit of the cell sample fromthe flow-through electroporation device; a flow channel intersecting andpositioned between the inlet channel and outlet channel; and two or moreelectrodes, wherein the two or more electrodes are (a) positioned in theflow channel between the intersection of the flow channel with the firstinlet channel and the intersection of the flow channel with the outletchannel, (b) in fluid communication with the cell sample in the flowchannel but are not in the flow path of the cell sample in the flowchannel, and (c) configured to apply an electric pulse or electricpulses to a cell sample.

In some aspects the automated instrument further comprises a scriptreadable by a processor wherein the script comprises commands forretrieving reagents in the reagent cartridge and commands forelectroporating cells in the electroporation device. In some aspects,the script readable by a processor comprises commands for performing oneor more additional processes in the automated instrument, and in someaspects, the script readable by a processor comprises commands forperforming all processes in the automated instrument.

In some aspects, the automated instrument further comprises a cellgrowth module, and in some aspects, the cell growth module comprises arotating growth vial. The automated instrument may also comprise afiltration module, where the filtration module comprises a hollow fiberfilter.

The automated instrument may also comprise a recovery module, where therecovery module comprises a cell growth module comprising a rotatinggrowth vial.

In some aspects the automated instrument further comprises a storagemodule, and/or a nucleic acid assembly module, where the nucleic acidassembly module is a Gibson assembly module or a Gap Repair module.

In many aspects, the automated instrument further comprises a processorto read the script.

Yet another embodiment of an automated instrument presented hereincomprises a reagent cartridge comprising a plurality of reagentreservoirs; a flow-through electroporation device, wherein theflow-through electroporation device comprises an inlet and inlet channelfor introduction of a cell sample to the flow-through electroporationdevice; an outlet and outlet channel for exit of the cell sample fromthe flow-through electroporation device; a constricted flow channelintersecting and positioned between the inlet channel and outletchannel; and two or more electrodes, wherein the two or more electrodesare (a) positioned in the flow channel between the intersection of theflow channel with the first inlet channel and the intersection of theflow channel with the outlet channel and on either side of theconstriction in the flow channel, (b) in fluid communication with thecell sample in the flow channel but are not in the flow path of the cellsample in the flow channel, and (c) configured to apply an electricpulse or electric pulses to a cell sample; and a script readable by aprocessor wherein the script comprises commands for retrieving reagentsin the reagent cartridge and commands for electroporating cells in theelectroporation device.

And yet another embodiment provides a kit for use in the automatedinstruments comprising a reagent cartridge, where the reagent cartridgefurther comprises reagents dispensed in one or more of the reagentreservoirs. In some aspects, the one or more reagent reservoirs withreagents dispensed therein is sealed. Also in some aspects, the reagentcartridge comprises a cover for the reagent cartridge.

In some aspects, the reagent cartridge of the kit comprises cellsdispensed in one or more reagent reservoirs, and in some aspects, thekit comprises an enzyme mix for a Gibson assembly reaction or a GapRepair reaction dispensed in one or more reagent reservoirs, and/ornucleic acid vectors and/or oligonucleotides dispensed in one or morereagent reservoirs. The kit may also comprise a rotating growth vialwith media and cells dispensed therein.

In addition, provided herein is an automated instrument comprising areagent cartridge comprising a plurality of reagent reservoirs; an(FTEP) device for introducing an nucleic acid into cells in a fluid,where the FTEP device comprises: at least one inlet and at least oneinlet channel for introducing a fluid comprising cells and nucleic acidto the FTEP device; an outlet and an outlet channel for removingtransformed cells and nucleic acid from the FTEP device; a flow channelpositioned between a first inlet channel and the outlet channel, whereinthe flow channel intersects with the first inlet channel and the outletchannel and wherein a portion of the flow channel narrows between theinlet channel intersection and the outlet channel intersection; and anelectrode positioned on either side of the flow channel and in directcontact with the fluid in the flow channel, the electrodes defining thenarrowed portion of the flow channel, and wherein the electrodes applyone or more electric pulses to the cells in the fluid as they passthrough the flow channel, thereby introducing the nucleic acid into thecells in the fluid.

In some aspects of this embodiment, the electrodes are positioned oneither side of the flow channel, are in direct contact with the fluid inthe flow channel and define the decrease in width of the flow channel.In some configurations of this aspect, the electrodes are between 10 μmto 5 mm apart, or between 25 μm to 2 mm apart.

In some aspects of these embodiments, the FTEP device is between 3 cm to15 cm in length, or between 4 cm to 12 cm in length, or from 4.5 cm to10 cm in length, or from 5 cm to 8 cm in length. In some aspects ofthese embodiments, this embodiment of the FTEP device is between 0.5 cmto 5 cm in width, or from 0.75 cm to 3 cm in width, or from 1 cm to 2.5cm in width, or from 1 cm to 1.5 cm in width. In some aspects of theseembodiments, the narrowest part of the channel width in the FTEP deviceis from 10 μM to 5 mm such that whatever cell type is being transformedwill not be physically contorted or “squeezed” by features of the FTEPdevice.

Also in some aspects of these embodiments, the flow rate in the FTEPranges from 0.1 ml to 5 ml per minute, or from 0.5 ml to 3 ml perminute, or from 1 ml to 2.5 ml per minute. In some aspects of theseembodiments the electrodes are configured to deliver 1-25 Kv/cm, or10-20 Kv/cm.

In some aspects of these embodiments, the FTEP device further comprisesone or more filters between the one or more inlet channels and theoutlet channel. In some aspects, there are two filters, one between theinlet channel and the narrowed portion of the flow channel, and onebetween the narrowed portion of the flow channel and the outlet channel.In some aspects of these embodiments, the filters are graduated in poresize with the larger pores proximal to the inlet chamber or outletchamber, and the small pores proximal to the narrowed portion of theflow channel. In some aspects, the small pores are the same size orlarger than the size of the narrowed portion of the flow channel. Insome aspects of these embodiments, the filter is formed separately fromthe body of the FTEP device and placed into the FTEP device as it isbeing assembled. Alternatively, in some aspects of these embodiments,the filter may be formed as part of and integral to the body of the FTEPdevice.

In some aspects of these embodiments, the FTEP device further comprisesa reservoir connected to the inlet for introducing the cells in fluidinto the FTEP device and a reservoir connected to the outlet forremoving transformed cells from the FTEP device, and in some aspects,the FTEP device comprises two inlets and two inlet channels and furthercomprises a reservoir connected to a second inlet for introducing thenucleic acid into the FTEP device. In some aspects the FTEP devicecomprises a reservoir connected to the inlet for introducing both thecells in fluid and the nucleic acid into the FTEP device and a reservoirconnected to the outlet for removing transformed cells from the FTEPdevice In some aspects of these embodiments, the reservoirs coupled tothe inlet(s) and outlet range in volume from 100 μL to 10 ml, or from0.5 ml to 7 ml, or from 1 ml to 5 ml.

In some aspects of these embodiments, the FTEP devices can provide acell transformation rate of 10³ to 10¹² cells per minute, or 10⁴ to 10¹⁰per minute, or 10⁵ to 10⁹ per minute, or 10⁶ to 10⁸ per minute.Typically, 10⁸ yeast cells may be transformed per minute, and 10¹⁰-10¹¹bacterial cells may be transformed per minute. In some aspects of theseembodiments, the transformation of cells results in at least 90% viablecells, or 95% viable cells, and up to 99% viable cells.

In some aspects of these embodiments, the FTEP device is manufactured byinjection molding from crystal styrene, cyclo-olefin polymer, orcyclo-olefin co-polymer, and in some aspects of this embodiment theelectrodes are fabricated from stainless steel. In some aspects of theseembodiments, the FTEP devices are fabricated as multiple FTEP devices inparallel on a single substrate where the FTEP devices are then separatedfor use.

In some embodiments of the automated multi-module cell processing systemof which the FTEP is a part, the nucleic acids in the one or morereceptacles comprise a vector backbone and an oligonucleotide, and theautomated instrument further comprises a nucleic acid assembly module.In some aspects, the nucleic acid assembly module comprises a magnet,and in some aspects, the nucleic acid assembly module is configured toperform nucleic acid assembly using a single, isothermal reaction. Inother aspects, the nucleic acid assembly module is configured to performan amplification and/or ligation method. In some aspects, the nucleicacid assembly module also comprises means for isolating, washing,concentrating, diluting and/or resuspending the assembled nucleic acids.

In some embodiments, the automated instrument comprising the FTEP mayfurther comprise a growth module configured to grow the cells, and insome implementations, the growth module measures optical density of thegrowing cells, either continuously or at intervals. In someimplementations, a processor controlling the instrument is configured toadjust growth conditions in the growth module such that the cells reacha target optical density at a time requested by a user. Further, in someembodiments, the user may be updated regarding growth process, e.g.through a user interface of the automated instrument or through aportable computing device application in communication with theautomated instrument.

In some embodiments, the automated instrument comprising the FTEP alsocomprises a reagent cartridge with one or more receptacles configured toreceive cells and one or more receptacles configured to receive nucleicacids. In some embodiments, the automated instrument comprising the FTEPalso comprises a reagent cartridge with one or more receptaclesconfigured to receive both cells and nucleic acids. Further, the reagentcartridge may also contain some or all reagents required for cellmanipulation following transformation, e.g., an antibiotic for selectionof transformed cell or an inducer for protein expression. In someimplementations, the reagents contained within the reagent cartridge arelocatable by a script read by the processor, and in someimplementations, the reagent cartridge includes reagents and is providedin a kit. In some embodiments, the FTEP device (e.g., transformationmodule) is contained within the reagent cartridge.

Some embodiments of the automated instrument further comprise afiltration module configured to exchange the liquid medium in which thecells are suspended and/or concentrate the cells. In specific aspects,the script comprises commands to alert a user that a target OD has beenreached by the cell growth module, and/or the script comprises commandsto adjust the growth temperature of cells to reach a target OD at atarget time.

In certain aspects, the nucleic acids are nucleic acids, and theinstrument further comprises a cell expression module, e.g., for theexpression of proteins encoded on the nucleic acids introduced to thetransformed cell populations.

In other aspects, the instrument may contain two or more electroporationdevices for performing two or more transformation events in a singleinstrument operation.

Other features, advantages, and aspects will be described below in moredetail.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of the specification, illustrate one or more embodiments and,together with the description, explain these embodiments. Theaccompanying drawings have not necessarily been drawn to scale. Anydimensions illustrated in the accompanying graphs and figures are forillustration purposes only and may or may not represent actual orpreferred values or dimensions. Where applicable, some or all featuresmay not be illustrated to assist in the description of underlyingfeatures. In the drawings:

FIG. 1 is a flow chart for an example method for automated introductionof nucleic acids.

FIGS. 2A and 2B depict side and front views of the automated instrumentfor introducing nucleic acids into cells. FIGS. 2C and 2D depict asecond example chassis of an automated instrument for introducingnucleic acids into cells.

FIG. 3 depicts an example combination nucleic acid assembly module andpurification module for use in an automated instrument.

FIG. 4A is an illustration of a top view of one embodiment of the FTEPdevices of the disclosure. FIG. 4B is an illustration of the top view ofa cross section of the embodiment of the device shown in FIG. 4A. FIG.4C is an illustration of a side view of a cross section of theembodiment of the device shown in FIGS. 4A and 4B. FIG. 4D is anillustration of a top view of another embodiment of the FTEP devices ofthe disclosure. FIG. 4E is an illustration of the top view of a crosssection of the embodiment of the device shown in FIG. 4D. FIG. 4F is anillustration of a side view of a cross section of the embodiment of thedevice shown in FIGS. 4D and 4E. FIG. 4G is an illustration of a topview of yet another embodiment of the FTEP devices of the disclosure.FIG. 4H is an illustration of the top view of a cross section of theembodiment of the device shown in FIG. 4G. FIG. 4I is an illustration ofa side view of a cross section of the embodiment of the device shown inFIGS. 4G and 4H.

FIG. 5A is an illustration of the top view of a cross section of afurther embodiment of the FTEP devices described herein with separateinlets for the cells and the nucleic acids. FIG. 5B is an illustrationof the top view of a cross section of the embodiment of the device shownin FIG. 5A. FIG. 5C is an illustration of a side view of a cross sectionof the embodiment of the device shown in FIG. 5B. FIG. 5D is anillustration of a side view of a cross section of a variation on theembodiment of the device shown in FIGS. 5A and 5B. FIG. 5E is anillustration of a side view of a cross section of another variation onthe embodiment of the device shown in FIGS. 5C and 5D.

FIG. 5F is an illustration of the top view of a cross section of yetanother embodiment of the FTEP devices of the disclosure where the FTEPcomprises two separate inlets for the cells and the nucleic acids. FIG.5G is an illustration of a top view of a cross section of the embodimentof the device shown in FIG. 5F. FIG. 5H is an illustration of a sideview of a cross section of the embodiment of the device shown in FIGS.5F and 5G.

FIG. 6 is an illustration of a top view of a cross section of yet anadditional embodiment of the FTEP devices of the disclosure, hereincluding flow focusing of fluid from the input channels.

FIG. 7A is an illustration of a top view of a cross section of a firstmultiplexed embodiment of the FTEP devices of the disclosure. FIG. 7B isan illustration of a top view of a cross section of a second multiplexedembodiment of the devices of the disclosure. FIG. 7C is an illustrationof a top view of a cross section of a third multiplexed embodiment ofthe devices of the disclosure. FIG. 7D is an illustration of a top viewof a cross section of a fourth multiplexed embodiment of the devices ofthe disclosure. FIG. 7E is an illustration of a top view of a crosssection of a fifth multiplexed embodiment of the devices of thedisclosure.

FIG. 8A is an illustration of a top view of yet another embodiment ofthe FTEP devices of the disclosure where the electrodes are placed oneither end of the narrowed region of the flow channel rather than oneither side and defining the narrowed region of the flow channel. FIG.8B is an illustration of the top view of a cross section of theembodiment of the device shown in FIG. 8A. FIG. 8C is an illustration ofa side view of a cross section of the embodiment of the device shown inFIGS. 8A and 8B. FIG. 8D is an illustration of a side view of a crosssection of the bottom half of the embodiment of the devices shown inFIGS. 8A, 8B and 8C. FIG. 8E is an illustration of a side view of across section of a variation of the embodiment of the devices shown inFIGS. 8A-8D where here the electrodes are positioned on the bottom ofthe FTEP device, on the opposite surface from the inlet and outlet. FIG.8F is an illustration of a top view of yet another embodiment of theFTEP devices of the disclosure. FIG. 8G an illustration of the top viewof a cross section of the embodiment of the device shown in FIG. 8F.FIG. 8H is an illustration of a side view of a cross section of onevariation of the embodiment of the device shown in FIGS. 8F and 8G.

FIG. 8I is an illustration of a top view of an embodiment of the FTEPdevices of the disclosure. FIG. 8J is an illustration of the top view ofa cross section of the embodiment of the device shown in FIG. 8I wherein this embodiment the FTEP device comprises a filter. FIG. 8K is anillustration of the top view of a cross section of a variation of theembodiment of the device shown in FIGS. 8I and 8J. FIG. 8L is anillustration of a side view of a cross section of the embodiment of thedevices shown in FIGS. 8I-8K. FIG. 8M is an illustration of a side viewof a cross section of the bottom half of the embodiment of the devicesshown in FIGS. 8I-8L. FIG. 8N is an illustration of a top view of yetanother embodiment of the FTEP devices of the disclosure. FIG. 8O is anillustration of the top view of a cross section of the embodiment of thedevice shown in FIG. 8N. FIG. 8P is an illustration of a side view of across section of the embodiment of the device of the disclosure shown inFIGS. 8N-8O. FIG. 8Q is an illustration of a side view of a crosssection of a variation on the embodiment of the device shown in FIGS.8N-8O. FIG. 8R is an illustration of a side view of a cross section ofanother variation on the embodiment of the device shown in FIGS. 8N-8Q.

FIG. 8S is an illustration of the top view of a cross section of yetanother embodiment of the FTEP devices of the disclosure. FIG. 8T is anillustration of the top view of a cross section of the embodiment of thedevice shown in FIG. 8S. FIG. 8U is an illustration of a side view of across section of the embodiment of the device shown in FIGS. 8S and 8T.

FIG. 9A is an illustration of a side view of a cross section of anotherembodiment of the FTEP devices of the disclosure. FIG. 9B is anillustration of the top view of a cross section of the embodiment of thedevice shown in FIG. 9A. FIG. 9C is an illustration of a top view of across section of an embodiment of an FTEP device with a flow focusingfeature.

FIGS. 10A through 10C are top perspective, bottom perspective, andbottom views, respectively, of a flow-through electroporation devicethat may be part of a stand-alone FTEP module or as one module in anautomated multi-module cell processing system. FIG. 10D shows scanningelectromicrographs of the FTEP units depicted in FIG. 10C. FIG. 10Eshows scanning electromicrographs of filters 1070 and 1502 depicted asblack bars in FIGS. 10B and 10C. FIG. 10F depicts (i) the electrodesbefore insertion into the FTEP device; (ii) an electrode; and (iii) theelectrode inserted into an electrode channel with the electrode andelectrode channel adjacent to the flow channel. FIG. 10G shows twoscanning electromicrographs of two different configurations of theaperture where the electrode channel meets the flow channel.

FIGS. 11A-11B depict an exploded view and a top view, respectively, ofan example wash cartridge for use in an automated instrument. FIGS.11C-11E depict an example reagent cartridge for use in an automatedinstrument.

FIGS. 12A-12C provide a functional block diagram and two perspectiveviews of an example filtration module for use in an automatedinstrument. FIG. 12D is a perspective view of an example filtercartridge for use in an automated instrument.

FIGS. 13A-13C depict example cell growth module components for use in anautomated instrument for introduction of nucleic acids.

FIG. 14 is an example control system for use in an automated instrument.

FIG. 15A is a flow diagram of a first example workflow for automatedintroduction of nucleic acids in an automated instrument. FIG. 15B is aflow diagram of a first example workflow for automated introduction ofnucleic acids in an instrument. FIG. 15C is a flow diagram of a secondexample workflow for automated introduction of nucleic acids withadditional protein expression and isolation.

FIG. 16 illustrates an example graphical user interface for providinginstructions to and receiving feedback from an instrument for automatedintroduction of nucleic acids instrument.

FIG. 17A is a functional block system diagram of another exampleembodiment of an automated instrument for the automated transformationof multiple cells. FIG. 17B is a functional block system diagram of yetanother example embodiment of an automated instrument for thetransformation of multiple cells.

FIG. 18A is a bar graph showing the results of electroporation of E.coli using a device of the disclosure and a comparator electroporationdevice. FIG. 18B is a bar graph showing uptake, cutting, and editingefficiencies of E. coli cells transformed via an FTEP as describedherein benchmarked against a comparator electroporation device.

FIG. 19 is a bar graph showing the results of electroporation of S.cerevisiae using an FTEP device of the disclosure and a comparatorelectroporation method.

FIG. 20 shows a graph of FTEP flow and pressure versus elapsed time(top), as well as a simple depiction of the pressure system and FTEP(bottom).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The description set forth below in connection with the appended drawingsis intended to be a description of various, illustrative embodiments ofthe disclosed subject matter. Specific features and functionalities aredescribed in connection with each illustrative embodiment; however, itwill be apparent to those skilled in the art that the disclosedembodiments may be practiced without each of those specific features andfunctionalities. Moreover, all of the functionalities described inconnection with one embodiment are intended to be applicable to theadditional embodiments described herein except where expressly stated orwhere the feature or function is incompatible with the additionalembodiments. For example, where a given feature or function is expresslydescribed in connection with one embodiment but not expressly mentionedin connection with an alternative embodiment, it should be understoodthat the feature or function may be deployed, utilized, or implementedin connection with the alternative embodiment unless the feature orfunction is incompatible with the alternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and sequencing technology,which are within the skill of those who practice in the art. Suchconventional techniques include synthesis, assembly, hybridization andligation of polynucleotides, and detection of hybridization using alabel. Specific illustrations of suitable techniques can be had byreference to the examples herein. However, other equivalent conventionalprocedures can, of course, also be used. Such conventional techniquesand descriptions can be found in standard laboratory manuals such asGreen, et al., Eds. (1999), Genome Analysis: A Laboratory Manual Series(Vols. I-IV); Weiner, Gabriel, Stephens, Eds. (2007), Genetic Variation:A Laboratory Manual; Dieffenbach, Dveksler, Eds. (2003), PCR Primer: ALaboratory Manual; Bowtell and Sambrook (2003), DNA Microarrays: AMolecular Cloning Manual; Mount (2004), Bioinformatics: Sequence andGenome Analysis; Sambrook and Russell (2006), Condensed Protocols fromMolecular Cloning: A Laboratory Manual; and Sambrook and Russell (2002),Molecular Cloning: A Laboratory Manual (all from Cold Spring HarborLaboratory Press); Stryer, L. (1995) Biochemistry (4th Ed.) W.H.Freeman, New York N.Y.; Gait, “Oligonucleotide Synthesis: A PracticalApproach” 1984, IRL Press, London; Nelson and Cox (2000), Lehninger,Principles of Biochemistry 3^(rd) Ed., W.H. Freeman Pub., New York,N.Y.; Berg et al. (2002) Biochemistry, 5^(th) Ed., W.H. Freeman Pub.,New York, N.Y.; Cell and Tissue Culture: Laboratory Procedures inBiotechnology (Doyle & Griffiths, eds., John Wiley & Sons 1998);Mammalian Chromosome Engineering—Methods and Protocols (G. Hadlaczky,ed., Humana Press 2011); Essential Stem Cell Methods, (Lanza andKlimanskaya, eds., Academic Press 2011), all of which are hereinincorporated in their entirety by reference for all purposes.CRISPR-specific techniques can be found in, e.g., Genome Editing andEngineering From TALENs and CRISPRs to Molecular Surgery, Appasani andChurch, 2018; and CRISPR: Methods and Protocols, Lindgren andCharpentier, 2015; both of which are herein incorporated in theirentirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “an oligo” refers toone or more oligos that serve the same function, to “the methods”includes reference to equivalent steps and methods known to thoseskilled in the art, and so forth. That is, unless expressly specifiedotherwise, as used herein the words “a,” “an,” “the” carry the meaningof “one or more.” Additionally, it is to be understood that terms suchas “left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”“outer” that may be used herein merely describe points of reference anddo not necessarily limit embodiments of the present disclosure to anyparticular orientation or configuration.

Furthermore, terms such as “first,” “second,” “third,” etc., merelyidentify one of a number of portions, components, steps, operations,functions, and/or points of reference as disclosed herein, and likewisedo not necessarily limit embodiments of the present disclosure to anyparticular configuration or orientation.

Furthermore, the terms “approximately,” “proximate,” “minor,” andsimilar terms generally refer to ranges that include the identifiedvalue within a margin of 20%, 10% or preferably 5% in certainembodiments, and any values therebetween.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs.

All publications (including patents, published applications, andnon-patent literature) mentioned herein are incorporated by referencefor all purposes, including but not limited to the purpose of describingand disclosing devices, systems, and methods that may be used ormodified in connection with the presently described methods, modules,instruments, and systems.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the disclosure. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the disclosure, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the disclosure.

Reference throughout the specification to “one embodiment” or “anembodiment” means that a particular feature, structure, orcharacteristic described in connection with an embodiment is included inat least one embodiment of the subject matter disclosed. Thus, theappearance of the phrases “in one embodiment” or “in an embodiment” invarious places throughout the specification is not necessarily referringto the same embodiment.

Further, the particular features, structures or characteristics may becombined in any suitable manner in one or more embodiments. Further, itis intended that embodiments of the disclosed subject matter covermodifications and variations thereof.

Introduction and Overview

The present disclosure provides automated instruments comprising FTEPdevices for the automated introduction of nucleic acids into livingcells. In some embodiments, the automated instruments include both anFTEP module and a nucleic acid assembly modules to introduction of anoligonucleotide or nucleic acid of interest into a vector backbone thatcontrols expression or other control of the oligonucleotide or nucleicacid. Each system described herein has advantages and challenges, andthe particular system that can be used in the inventions of thedisclosure can be selected for the particular application, as will beapparent to one of ordinary skill in the art upon reading the presentdisclosure.

The cells that can be transformed or transfected using the automatedinstrument comprising the FTEP devices include any prokaryotic, archaealor eukaryotic cell. For example, prokaryotic cells for use with thepresent illustrative embodiments can be gram positive bacterial cells,e.g., Bacillus subtilis, or gram negative bacterial cells, e.g., E. colicells. Eukaryotic cells for use with the automated instruments of theillustrative embodiments include any plant cells and any animal cells,e.g. fungal cells, insect cells, amphibian cells, nematode cells, ormammalian cells.

FIG. 1 is a flow chart for an example method 100 for automatedintroduction of nucleic acids. In a first step 102, cells of interestare transferred to a growth module (as described in detail below), wherethe cells are grown to a desired optical density 104. The cells are thentransferred from the growth module to a filtration module 106, whereinthe cells are concentrated, and in certain embodiments, concurrently thecells are rendered electrocompetent 108. Optionally in a parallelprocess A, nucleic acids (such as, e.g., a vector backbone and anexpression cassette) are transferred 110 to a nucleic acid assemblymodule (also as described in detail below) where assembly of the, e.g.,expression cassette into the vector backbone is performed 112. Once thenucleic acid assembly has been accomplished, the assembled nucleic acidsare transferred 114 to a purification module, where the nucleic acidsare, e.g., de-salted, washed, and/or sorted (e.g., where assemblednucleic acids are separated from unassembled vectors and expressioncassettes). After purification, the assembled nucleic acids are filteredand eluted 116. At this point, the concentrated and electrocompetentcells and the assembled nucleic acids are transferred 118 to the FTEPdevice, where the cells are transformed or transfected 120 with theassembled nucleic acids. Following transformation, the cells may betransferred 122 to a second growth module where the cells are allowed torecover. In the second growth module, there may be selective medium toselect for transformed cells, or the cells may be subjected to, e.g.,cell editing or protein expression. Next, the cells may be moved 124 toa storage, isolation, and/or processing module. In some aspects, thecells may go through another cycle of processing (e.g., repeating steps108, 118, 120, 122, 124 with another set of assembled nucleic acids viaprocess A), or the cells may be removed and used in further experimentsor analysis 126.

Instrument Architecture

FIGS. 2A through 2D illustrate example chassis 200 and 230 for use indesktop versions of an automated multi-module cell processinginstrument. For example, the chassis 200 and 230 may have a width ofabout 24-48 inches, a height of about 24-48 inches and a depth of about24-48 inches. Each of the chassis 200 and 230 may be designed to holdmultiple modules and disposable supplies used in automated cellprocessing. Further, each chassis 200 and 230 may mount a robotichandling system for moving materials between modules.

FIGS. 2A and 2B depict a first example chassis 200 of an automatedmulti-module cell processing instrument. As illustrated, the chassis 200includes a cover 202 having a handle 204 and hinges 206 a-206 c forlifting the cover 202 and accessing an interior of the chassis 200. Acooling grate 214 may allow for air flow via an internal fan (notshown). Further, the chassis 200 is lifted by adjustable feet 220. Thefeet 220 a-220 c, for example, may provide additional air flow beneaththe chassis 200. A control button 216, in some embodiments, allows forsingle-button automated start and/or stop of cell processing within thechassis 200.

Inside the chassis 200, in some implementations, a robotic handlingsystem 208 is disposed along a gantry 210 s or 210 b above materialscartridges 212 a, 212 b. Control circuitry, liquid handling tubes, airpump controls, valves, thermal units (e.g., heating and cooling units)and other control mechanisms, in some embodiments, are disposed below adeck of the chassis 200, in a control box region 218.

Although not illustrated, in some embodiments a display screen may bepositioned upon a front face of the chassis 200, for example covering aportion of the cover 202. The display screen may provide information tothe user regarding a processing status of the automated multi-modulecell processing instrument. In another example, the display screen mayaccept inputs from the user for conducting the cell processing.

FIGS. 2C and 2D depict a second example chassis 230 of an automatedmulti-module cell processing instrument. The chassis 230, asillustrated, includes a transparent door 232 with a hinge 234. Forexample, the door may swing to the left of the page to provide access toa work area of the chassis. The user, for example, may open thetransparent door 232 to load supplies, such as reagent cartridges andwash cartridges, into the chassis 230.

In some embodiments, a front face of the chassis 230 further includes adisplay (e.g., touch screen display device) 236 illustrated to the rightof the door 232. The display 236 may provide information to the userregarding a processing status of the automated multi-module cellprocessing instrument. In another example, the display 236 may acceptinputs from the user, e.g., for pausing or conducting the cellprocessing.

An air grate 238 on a right face of the chassis 230 may provide for airflow within a work area (e.g., above the deck) of the chassis 230 (e.g.,above a deck). A second air grate 240 on a left of the chassis 230 mayprovide for air flow within a control box region 242 (e.g., below thedeck) of the chassis 230. Although not illustrated, in some embodiments,feet such as the feet 220 a-220 c of the chassis 200 may raise thechassis 230 above a work surface, providing for further air flow.

Inside the chassis 230, in some implementations, a robotic handlingsystem 248 is disposed along a gantry 250 above cartridges 252 a, 252 b,material supplies 254 a, 254 b (e.g., pipette tips and filters), andmodules (e.g., dual growth vials, FTEP device, nucleic acid assemblymodule (not shown)). Control circuitry, liquid handling tubes, air pumpcontrols, valves, and other control mechanisms, in some embodiments, aredisposed below a deck of the chassis 230, in the control box region 242.

In some embodiments, a liquid waste unit 246 is mounted to the leftexterior wall of the chassis 230. The liquid waste unit 246, forexample, may be mounted externally to the chassis 230 to avoid potentialcontamination and to ensure prompt emptying and replacement of theliquid waste unit 246.

Nucleic Acid Assembly Module

Certain embodiments of the automated instruments of the presentdisclosure include a nucleic acid assembly module instrument. Thenucleic acid assembly module is configured to accept and assemble thenucleic acids necessary to facilitate the desired genome manipulations.The nucleic acid assembly module may also be configured to accept theappropriate vector backbone for vector assembly and subsequentelectroporation into the cells of interest.

In general, the term “vector” refers to a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked.Vectors include, but are not limited to, nucleic acid molecules that aresingle-stranded, double-stranded, or partially double-stranded; nucleicacid molecules that include one or more free ends, no free ends (e.g.circular); nucleic acid molecules that include DNA, RNA, or both; andother varieties of polynucleotides known in the art. One type of vectoris a “plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,where virally-derived DNA or RNA sequences are present in the vector forpackaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses). Viral vectors also include polynucleotidescarried by a virus for transfection into a host cell. Certain vectorsare capable of autonomous replication in a host cell into which they areintroduced (e.g. bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors.” Commonexpression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. Additional vectors include fosmids, phagemids, andsynthetic chromosomes.

Recombinant expression vectors can include a nucleic acid in a formsuitable for transformation, and for some nucleic acid sequences,translation and expression of the nucleic acid in a host cell, whichmeans that the recombinant expression vectors include one or moreregulatory elements—which may be selected on the basis of the host cellsto be used for expression—that are operatively-linked to the nucleicacid sequence to be expressed. Within a recombinant expression vector,“operably linked” is intended to mean that the nucleotide sequence ofinterest is linked to the regulatory element(s) in a manner that allowsfor transcription, and for some nucleic acid sequences, translation andexpression of the nucleotide sequence (e.g. in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell). Appropriate recombination and cloningmethods are disclosed in U.S. patent application Ser. No. 10/815,730,entitled “Recombinational Cloning Using Nucleic Acids HavingRecombination Sites” published Sep. 2, 2004 as US 2004-0171156 A1, thecontents of which are herein incorporated by reference in their entiretyfor all purposes.

In some embodiments, a regulatory element is operably linked to one ormore elements of a targetable nuclease system so as to drivetranscription, and for some nucleic acid sequences, translation andexpression of the one or more components of the targetable nucleasesystem.

In addition, the polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcells, such as prokaryotic or eukaryotic cells. Eukaryotic cells can beyeast, fungi, algae, plant, animal, or human cells. Eukaryotic cells maybe those of or derived from a particular organism, such as a mammal,including but not limited to human, mouse, rat, rabbit, dog, ornon-human mammal including non-human primate. In addition oralternatively, a vector may include a regulatory element operably likedto a polynucleotide sequence, which, when transcribed, forms a guideRNA.

The nucleic acid assembly module can be configured to perform a widevariety of different nucleic acid assembly techniques in an automatedfashion. Nucleic acid assembly techniques that can be performed in thenucleic acid assembly module of the disclosed automated instrumentsinclude, but are not limited to, those assembly methods that userestriction endonucleases, including PCR, BioBrick assembly (U.S. Pat.No. 9,361,427 to Hillson entitled “Scar-less Multi-part DNA AssemblyDesign,” issued Jun. 7, 2016), Type IIS cloning (e.g., GoldenGateassembly; European Patent Application Publication EP 2 395 087 A1 toWeber et al. entitled “System and Method of Modular Cloning,” filed Jul.6, 2010), and Ligase Cycling Reaction (de Kok S, ACS Synth Biol.,3(2):97-106 (2014); Engler, et al., PLoS One, 3(11):e3647 (2008); U.S.Pat. No. 6,143,527 to Pachuk et al. entitled “Chain Reaction CloningUsing a Bridging Oligonucleotide and DNA Ligase,” issued Nov. 7, 2000).In other embodiments, the nucleic acid assembly techniques performed bythe disclosed automated instruments are based on overlaps betweenadjacent parts of the nucleic acids, such as Gibson Assembly®, CPEC,SLIC, Ligase Cycling etc. Additional assembly methods include gap repairin yeast (Bessa, Yeast, 29(10):419-23 (2012)), gateway cloning (Ohtsuka,Curr Pharm Biotechnol, 10(2):244-51(2009); U.S. Pat. No. 5,888,732 toHartley et al., entitled “Recombinational Cloning Using EngineeredRecombination Sites,” issued Mar. 30, 1999; U.S. Pat. No. 6,277,608 toHartley et al. entitled “Recominational Cloning Using Nucleic AcidsHaving Recombination Sites,” issued Aug. 21, 2001), andtopoisomerase-mediated cloning (Udo, PLoS One, 10(9):e0139349 (2015);U.S. Pat. No. 6,916,632 B2 to Chestnut et al. entitled “Methods andReagents for Molecular Cloning,” issued Jul. 12, 2005). These and othernucleic acid assembly techniques are described, e.g., in Sands andBrent, Curr Protoc Mol Biol., 113:3.26.1-3.26.20 (2016); Casini et al.,Nat Rev Mol Cell Biol., (9):568-76 (2015); Patron, Curr Opinion PlantBiol., 19:14-9 (2014)).

The nucleic acid assembly module is temperature controlled dependingupon the type of nucleic acid assembly used in the automated instrument.For example, when PCR is utilized in the nucleic acid assembly module,the module will have a thermocycling capability allowing thetemperatures to cycle between denaturation, annealing and extension.When single temperature assembly methods are utilized in the nucleicacid assembly module, the module will have the ability to reach and holdat the temperature that optimizes the specific assembly process beingperformed. These temperatures and the duration for maintaining thesetemperatures can be determined by a preprogrammed set of parametersexecuted by a script, or manually controlled by the user using theprocessing system of the automated instrument.

In one embodiment, the nucleic acid assembly module is a module toperform assembly using a single, isothermal reaction, such as thatillustrated in FIG. 3. The isothermal assembly module is configured toperform the molecular cloning method using the single, isothermalreaction. Certain isothermal assembly methods can combine simultaneouslyup to 15 nucleic acid fragments based on sequence identity. The assemblymethod provides, in some embodiments, nucleic acids to be assembledwhich include an approximate 20-40 base overlap with adjacent nucleicacid fragments. The fragments are mixed with a cocktail of threeenzymes—an exonuclease, a polymerase, and a ligase-along with buffercomponents. Because the process is isothermal and can be performed in a1-step or 2-step method using a single reaction vessel, isothermalassembly reactions are ideal for use in an automated instrument. The1-step method allows for the assembly of up to five different fragmentsusing a single step isothermal process. The fragments and the master mixof enzymes are combined and incubated at 50° C. for up to one hour. Forthe creation of more complex constructs with up to fifteen fragments orfor incorporating fragments from 100 bp up to 10 kb, typically the2-step is used, where the 2-step reaction requires two separateadditions of master mix; one for the exonuclease and annealing step anda second for the polymerase and ligation steps.

FIG. 3 illustrates an example nucleic acid assembly module 300 withintegrated purification. The nucleic acid assembly module 300 includes achamber 302 having an access gasket 304 for transferring liquids to andfrom the nucleic acid assembly module 300 (e.g., via a pipette orsipper). In some embodiments, the access gasket 304 is connected to areplaceable vial which is positioned within the chamber 302. Forexample, a user or robotic manipulation system may place the vial withinthe nucleic acid assembly module 300 for processing.

The chamber 302 shares a housing 306 with a resistive heater 308. Once asample has been introduced to the chamber 302 of the nucleic acidassembly module 300, the resistive heater 308 may be used to heat thecontents of the chamber 302 to a desired temperature. Thermal rampingmay be set based upon the contents of the chamber 302 (e.g., thematerials supplied through the access gasket 304 via pipettor or sipperunit of the robotic manipulation system). The processing system of theautomated instrument may determine the target temperature and thermalramping plan. The thermal ramping and target temperature may becontrolled through monitoring a thermal sensor such as a thermistor 310included within the housing 306. In a particular embodiment, theresistive heater 308 is designed to maintain a temperature within thehousing 306 of between 20° and 80° C., between 25° and 75° C., between37° and 65° C., between 40° and 60° C., between 45° and 55° C. orpreferably about 50° C.

Purification Module

In some embodiments, when a nucleic acid assembly module is included inthe automated instrument, the instrument also can include a purificationmodule to remove unwanted components of the nucleic acid assemblymixture (e.g., salts, minerals) and, in certain embodiments, concentratethe assembled nucleic acids. Examples of methods for exchanging theliquid following nucleic acid assembly include magnetic beads (e.g.,SPRI or Dynal (Dynabeads) by Invitrogen Corp. of Carlsbad, Calif.),silica beads, silica spin columns, glass beads, precipitation (e.g.,using ethanol or isopropanol), alkaline lysis, osmotic purification,extraction with butanol, membrane-based separation techniques,filtration etc.

In one aspect, the purification module provides filtration, e.g.,ultrafiltration. For example, a range of microconcentrators fitted withanisotropic, hydrophilic-generated cellulose membranes of varyingporosities is available. In another example, the purification andconcentration involves contacting a liquid sample including theassembled nucleic acids and an ionic salt with an ion exchangerincluding an insoluble phosphate salt, removing the liquid, and elutingthe nucleic acid from the ion exchanger.

In a specific aspect of the purification module, SPRI beads can be usedwhere 0.6-2.0× volumes of SPRI beads can be added to the nucleic acidassembly. The nucleic acid assembly product becomes bound to the SPRIbeads, and the SPRI beads are pelleted by automatically positioning amagnet close to the tube, vessel, or chamber harboring the pellet. Forexample, 0.6-2.0× volumes of SPRI beads can be added to the nucleic acidassembly. The SPRI beads, for example, may be washed with ethanol, andthe bound nucleic acid assembly product is eluted, e.g., in water, Trisbuffer, or 10% glycerol.

In a specific aspect, a magnet is coupled to a linear actuator thatpositions the magnet. In some implementations, the nucleic acid assemblymodule is a combination assembly and purification module designed forintegrated assembly and purification. For example, as discussed above inrelation to the nucleic acid assembly module depicted in FIG. 3, oncesufficient time has elapsed for the nucleic acid assembly reaction totake place, the contents of the chamber 302 (e.g., the nucleic acidassembly reagents and nucleic acids), in some embodiments, are combinedwith magnetic beads (not shown) to activate the purification process.The SPRI beads in buffer are delivered to the contents of the nucleicacid assembly module, for example, by a robotic handling system.Thereafter, a solenoid 312, in some embodiments, is actuated by a magnetto excite the magnetic beads contained within the chamber 302. Thesolenoid, in a particular example, may impart between a 2 pound magneticpull force and a 5 pound pull force, or approximately a 4 pound magneticpull force to the magnetic beads within the chamber 302. The contents ofthe chamber 302 may be incubated for sufficient time for the assembledvector and oligonucleotides to bind to the magnetic beads.

After binding, in some implementations, the bound nucleic acid assemblymix (e.g., isothermal nucleic acid assembly reagents+assembled vectorand oligonucleotides) is removed from the nucleic acid assembly moduleand the nucleic acids attached to the beads are washed one to severaltimes with 80% ethanol. Once washed, the nucleic acids attached to thebeads are eluted into buffer and are transferred to the transformationmodule. That is, in some embodiments, the nucleic acid assembly moduleand purification module are combined.

In some implementations, a vial is locked in position in the chamber 302for processing. For example, a user may press the vial beyond a detentin the chamber 302 designed to retain the vial upon engagement with apipettor or sipper. In another example, the user may twist the vial intoposition, thus engaging a protrusion to a corresponding channel andbarring upward movement. A position sensor (not illustrated) may ensureretraction of the vial. The position sensor, in a particular embodiment,is a magnetic sensor detecting engagement between a portion of thechamber 302 and the vial. In other embodiments, the position sensor isan optical sensor detecting presence of the vial at a retractedposition. In embodiments using a channel and protrusion, a mechanicswitch pressed down by the protrusion may detect engagement of the vial.

Growth Module

As the nucleic acids are being assembled, the cells may be grown inpreparation for transformation/transfection. Cell growth can bemonitored by optical density (e.g., at OD 600 nm) that is measured in agrowth module, and a feedback loop is used to adjust the cell growth soas to reach a target OD at a target time. Other measures of cell densityand physiological state that can be measured include but are not limitedto, pH, dissolved oxygen, released enzymes, acoustic properties, andelectrical properties.

In some aspects, the growth module includes a culture tube in a shakeror vortexer that is interrogated by a spectrophotometer or fluorimeter.The shaker or vortexer can heat or cool the cells and cell growth ismonitored by real-time absorbance or fluorescence measurements. In oneaspect, the cells are grown at 25° C.-40° C. to an OD600 absorbance of1-10 ODs. The cells may also be grown at temperature ranges from 25°C.-35° C., 25° C.-30° C., 30° C.-40° C., 30° C.-35° C., 35° C.-40° C.,40° C.-50° C., 40° C.-45° C. or 44° C.-50° C. In another aspect, thecells are induced by heating at 42° C.-50° C. or by adding an inducingagent. The cells may also be induced by heating at ranges from 42°C.-46° C., 42° C.-44° C., 44° C.-46° C., 44° C.-48° C., 46° C.-48° C.,46° C.-50° C., or 48° C.-50° C. In some aspects, the cells are cooled to0° C.-10° C. after induction. The cells may also be cooled totemperature ranges of 0° C.-5° C., 0° C.-2° C., 2° C.-4° C., 4° C.-6°C., 6° C.-8° C., 8° C.-10° C., or 5° C.-10° C. after induction.

FIG. 13A shows one embodiment of a rotating growth vial 1300 for usewith a cell growth device, such as cell growth device 1350 illustratedin FIGS. 13B-C. The rotating growth vial 1300, in some implementations,is a transparent container having an open end 1304 for receiving liquidmedia and cells, a central vial region 1306 that defines the primarycontainer for growing cells, a tapered-to-narrowed region 1318 definingat least one light path 1308, 1310, a closed end 1316, and a driveengagement mechanism 1312. The rotating growth vial 1300 may have acentral longitudinal axis 1320 around which the vial 1300 rotates, andthe light paths 1308, 1310 may be generally perpendicular to thelongitudinal axis of the vial. In some examples, first light path 1310may be positioned in the lower narrowed portion of thetapered-to-narrowed region 1318. The drive engagement mechanism 1312, insome implementations, engages with a drive mechanism (e.g., actuator,motor (not shown)) to rotate the vial 1300. The actuator may include adrive shaft 1374 for a drive motor (not shown).

In some embodiments, the rotating growth vial 1300 includes a secondlight path 1308, for example, in the upper tapered region of thetapered-to-narrowed region 1318. In some examples, the walls definingthe upper tapered region of the tapered-to-narrowed region 1318 for thesecond light path 1308 may be disposed at a wider angle relative to thelongitudinal axis 1320 than the walls defining the lower narrowedportion of the tapered-to-narrowed region 1310 for the first light path1310. Both light paths 1308, 1310, for example, may be positioned in aregion of the rotating growth vial 1300 that is constantly filled withthe cell culture (cells+growth media), and is not affected by therotational speed of the growth vial 1300. As illustrated, the secondlight path 1308 is shorter than the first light path 1310 allowing forsensitive measurement of optical density (OD) values when the OD valuesof the cell culture in the vial are at a high level (e.g., later in thecell growth process), whereas the first light path 1310 allows forsensitive measurement of OD values when the OD values of the cellculture in the vial are at a lower level (e.g., earlier in the cellgrowth process).

The rotating growth vial 1300 may be reusable, or preferably, therotating growth vial is consumable. In some embodiments, the rotatinggrowth vial 1300 is consumable and can be presented to the userpre-filled with growth medium, where the vial 1300 is sealed at the openend 1304 with a foil seal. A medium-filled rotating growth vial packagedin such a manner may be part of a kit for use with a stand-alone cellgrowth device or with a cell growth module that is part of an automatedinstrument. To introduce cells into the vial, a user need only pipetteup a desired volume of cells and use the pipette tip to punch throughthe foil seal of the vial 1300. Alternatively, of course, an automatedinstrument may transfer cells from, e.g., a reagent cartridge, to thegrowth vial. The growth medium may be provided in the growth vial or mayalso be transferred from a reagent cartridge to the growth vial beforethe addition of cells. Open end 1304 may include an extended lip 1302 tooverlap and engage with the cell growth device 1350 (FIG. 13B). Inautomated instruments, the rotating growth vial 1300 may be tagged witha barcode or other identifying means that can be read by a scanner orcamera that is part of the processing system 1410 as illustrated in FIG.14.

In some implementations, the volume of the rotating growth vial 1300 andthe volume of the cell culture (including growth medium) may varygreatly, but the volume of the rotating growth vial 1300 should be largeenough for the cell culture in the growth vial 1300 to get properaeration while the vial 1300 is rotating. In practice, the volume of therotating growth vial 1300 may range from 1-250 ml, 2-100 ml, from 5-80ml, 10-50 ml, or from 12-35 ml. Likewise, the volume of the cell culture(cells+growth media) should be appropriate to allow proper aeration inthe rotating growth vial 1300. Thus, the volume of the cell cultureshould be approximately 10-85% of the volume of the growth vial 800, or15-80% of the volume of the growth vial, or 20-70%, 30-60%, or 40-50% ofthe volume of the growth vial. In one example, for a 35 ml growth vial1300, the volume of the cell culture would be from about 4 ml to about27 ml.

The rotating growth vial 1300, in some embodiments, is fabricated from abio-compatible transparent material—or at least the portion of the vial1300 including the light path(s) is transparent. Additionally, materialfrom which the rotating growth vial 1300 is fabricated should be able tobe cooled to about 0° C. or lower and heated to about 75° C. or higher,such as about 2° C. or to about 70° C., about 4° C. or to about 60° C.,or about 4° C. or to about 55° C. to accommodate both temperature-basedcell assays and long-term storage at low temperatures. Further, thematerial that is used to fabricate the vial is preferably able towithstand temperatures up to 55° C. without deformation while spinning.Suitable materials include glass, polyvinyl chloride, polyethylene,polyamide, polyethylene, polypropylene, polycarbonate, poly(methylmethacrylate) (PMMA), polysulfone, polyurethane, and co-polymers ofthese and other polymers. Preferred materials include polypropylene,polycarbonate, or polystyrene. In some embodiments, the rotating growthvial 800 is inexpensively fabricated by, e.g., injection molding orextrusion.

FIGS. 13 B-C illustrate views of an example cell growth device 1350 thatreceives the rotating growth vial 1300. In some embodiments, the cellgrowth device 1350 rotates to heat or cool the cells or cell growthwithin the vial 1300 to a predetermined temperature range. In someimplementations, the rotating growth vial 1300 can be positioned insidea main housing 1352 with the extended lip 1302 of the vial 1300extending past an upper surface of the main housing 1352. In someaspects, the extended lip 1302 provides a grasping surface for a userinserting or withdrawing the vial 1300 from the main housing 1352 of thedevice 1350. Additionally, when fully inserted into the main housing1352, a lower surface of the extended lip 1302 abuts an upper surface ofthe main housing 1352. In some examples, the main housing 1352 of thecell growth device 1350 is sized such that outer surfaces of therotating growth vial 1300 abut inner surfaces of the main housing 1352thereby securing the vial 1300 within the main housing 1352. In someimplementations, the cell growth device 1350 can include end housings1354 disposed on each side of the main housing 1354 and a lower housing1356 disposed at a lower end of the main housing 1352. In some examples,the lower housing 1356 may include flanges 1358 that can be used toattach the cell growth device 1350 to a temperature control (e.g,heating/cooling) mechanism or other structure such as a chassis of anautomated cell processing system.

As shown in FIG. 13C, the cell growth device 1350, in someimplementations, can include an upper bearing 1360 and lower bearing1362 positioned in main housing 1352 that support the vertical load of arotating growth vial 1300 that has been inserted into the main housing1352. In some examples, the cell growth device 1350 may also include aprimary optical port 1366 and a secondary optical port 1368 that arealigned with the first light path 1310 and second light path 1308 of thevial 1300 when inserted into the main housing 1352. In some examples,the primary and secondary optical ports 1366, 1368 are gaps, openings,or portions of the main housing constructed from transparent materialsthat allow light to pass through the vial 1300 to perform cell growth ODmeasurements. In addition to the optical ports 1366, 1368, the cellgrowth device 1350 may include an emission board 1370 that provides oneor more illumination sources for the light path(s), and detector board1372 to detect the light after the light travels through the cellculture liquid in the rotating growth vial 1300. In one example, theillumination sources disposed on the emission board 1370 may includelight emission diodes (LEDs) or photodiodes that provide illumination atone or more target wavelengths commensurate with the growth mediatypically used in cell culture (whether, e.g., mammalian cells,bacterial cells, animal cells, yeast cells).

In some implementations, the emission board 1370 and/or detector board1372 are communicatively coupled through a wired or wireless connectionto a processing system (e.g., processing system 126, 1720, 1810) thatcontrols the wavelength of light output by the emission board 1370 andreceives and processes the illumination sensed at the detector board1372. The remotely controllable emission board 1370 and detector board1372, in some aspects, provide for conducting automated OD measurementsduring the course of cell growth. For example, the processing system126, 1720 may control the periodicity with which OD measurements areperformed, which may be at predetermined intervals or in response to auser request Further, the processing system 126, 1720 can use the sensordata received from the detector board 1372 to perform real-time ODmeasurements and adjust cell growth conditions (e.g., temperature,speed/direction of rotation).

In some embodiments, the lower housing 1356 may contain a drive motor(not shown) that generates rotational motion that causes the rotatinggrowth vial 1300 to spin within the cell growth device 1350. In someimplementations, the drive motor may include a drive shaft 1374 thatengages a lower end of the rotating growth vial 1300. The drive motorthat generates rotational motion for the rotating growth vial 1300, insome embodiments, is a brushless DC type drive motor with built-in drivecontrols that can be configured to maintain a constant revolution perminute (RPM) between 0 and about 3000 RPM. Alternatively, other motortypes such as a stepper, servo, or brushed DC motors can be used.Optionally, the drive motor may also have direction control to allowreversing of the rotational direction, and a tachometer to sense andreport actual RPM. In other examples, the drive motor can generateoscillating motion by reversing the direction of rotation at apredetermined frequency. In one example, the vial 1300 is rotated ineach direction for one second at a speed of 350 RPM. The drive motor, insome implementations, is communicatively coupled through a wired orwireless communication network to a processing system (e.g., processingsystem 126, 1720) that is configured to control the operation of thedrive motor, which can include executing protocols programmed into theprocessor and/or provided by user input, for example as described inrelation to module controller 1430 of FIG. 14. For example, and thedrive motor can be configured to vary the speed and/or rotationaldirection of the vial 1300 to cause axial precession of the cell culturethereby enhancing mixing in order to prevent cell aggregation andincrease aeration. In some examples, the speed or direction of rotationof the drive motor may be varied based on optical density sensor datareceived from the detector board 1372.

In some embodiments, main housing 1352, end housings 1354 and lowerhousing 1356 of the cell growth device 1350 may be fabricated from arobust material including aluminum, stainless steel, and other thermallyconductive materials, including plastics. These structures or portionsthereof can be created through various techniques, e.g., metalfabrication, injection molding, creation of structural layers that arefused, etc. While in some examples the rotating growth vial 1300 isreusable, in other embodiments, the vial 1300 is preferably isconsumable. The other components of the cell growth device 1350, in someaspects, are preferably reusable and can function as a stand-alonebenchtop device or as a module in an automated instrument.

In some implementations, the processing system that is communicativelycoupled to the cell growth module may be programmed with information tobe used as a “blank” or control for the growing cell culture. A “blank”or control, in some examples, is a vessel containing cell growth mediumonly, which yields 100% transmittance and 0 OD, while the cell samplesdeflect light rays and will have a lower percentage transmittance andhigher OD. As the cells grow in the media and become denser,transmittance decreases and OD increases. The processor of the cellgrowth module, in some implementations, may be programmed to usewavelength values for blanks commensurate with the growth mediatypically used in cell culture (whether, e.g., mammalian cells,bacterial cells, animal cells, yeast cells). Alternatively, a secondspectrophotometer and vessel may be included in the cell growth module,where the second spectrophotometer is used to read a blank at designatedintervals.

Cell Enrichment Module

To reduce background of cells that have not received a nucleic acid ofinterest, the instrument may comprise a cell enrichment module may alsoallow a selection process to increase the overall number of transformedcells in a cell populations created using thetransformation/transfection systems of the invention. In certainaspects, the cell enrichment module may be integrated with the cellgrowth module.

In some embodiments, enriching the sample includes one or more ofincreasing the overall percentage of cells of interest in the sample anddepleting cells not of interest in the sample.

For example, the introduced nucleic acid can include a gene, whichconfers antibiotic resistance or another selectable marker. Suitableantibiotic resistance genes include, but are not limited to, genes suchas ampicillin-resistance gene, tetracycline-resistance gene,kanamycin-resistance gene, neomycin-resistance gene,canavanine-resistance gene, blasticidin-resistance gene,hygromycin-resistance gene, puromycin-resistance gene, andchloramphenicol-resistance gene. In some embodiments, removing dead cellbackground is aided using lytic enhancers such as detergents, osmoticstress, temperature, enzymes, proteases, bacteriophage, reducing agents,or chaotropes. In other embodiments, cell removal and/or media exchangeis used to reduce dead cell background.

Cell Wash and/or Concentration Module

The cell wash and/or concentration module can utilize any method forexchanging the liquids in the cell environment, and may concentrate thecells or allow them to remain in essentially the same or greater volumeof liquid as used in the nucleic acid assembly module. Further, in someaspects, the processes performed in the cell wash module also render thecells electrocompetent, by, e.g., use of glycerol in the wash.

Numerous different methods can be used to wash the cells, includingdensity gradient purification, dialysis, ion exchange columns,filtration, centrifugation, dilution, and the use of beads forpurification.

In some aspects, the cell wash and/or concentration module utilizes acentrifugation device. In other aspects, the cell wash and/orconcentration module utilizes a filtration module. In yet other aspects,beads are coupled to moieties that bind to the cell surface. Thesemoieties include but are not limited to antibodies, lectins, wheat germagglutinin, mutated lysozymes, and ligands.

In other aspects, the cells are engineered to be magnetized, allowingmagnets to pellet the cells after wash steps. The mechanism of cellmagnetization can include but is not limited to ferritin proteinexpression.

The cell wash and/or concentration module, in some implementations, is afiltration module. Turning to FIG. 12A, a block diagram illustratesexample functional units of a filtration module 1200. In someimplementations, a main control 1202 of the filtration module 1200includes a first liquid pump 1204 b to intake wash fluid 1206 and asecond liquid pump 1204 b to remove liquid waste to a liquid wastemodule 1208 (e.g., such as the liquid waste module 1728 of FIGS. 17A and17B). A flow sensor 1212 may be disposed on a connector 1214 to theliquid waste module 1208 to monitor release of liquid waste from thefiltration module. A valve 1216 (a three-way valve as illustrated) maybe disposed on a connector 1218 to the wash fluid in wash cartridge 1210to selectively connect the wash fluid and the filtration module 1200.

The filtration module 1200, in some implementations, includes a filtermanifold 1220 for filtering and concentrating a cell sample. The filtermanifold 1220 may include one or more temperature sensor(s) 1222 andpressure sensor (s) 1224 to monitor flow and temperature of the washfluid and/or liquid waste. The sensors 1222, 1224, in some embodiments,are monitored and analyzed by a processing system of the automatedmulti-mode cell processing system, such as the processing system 1410 ofFIG. 14. The filter manifold 1220 may include one or more valves 1226 bfor directing flow of the wash fluid and/or liquid waste. The processingsystem of the automated multi-mode cell processing instrument, forexample, may actuate the valves according to a set of instructions fordirecting filtration by the filtration module 1200.

The filtration module 1200 includes at least one filter 1230. Examplesof filters suitable for use in the filtration module 1200 includemembrane filters, ceramic filters and metal filters. The filter may beused in any shape; the filter may for example be cylindrical oressentially flat. The filter selected for a given operation or a givenworkflow, in some embodiments, depends upon the type of workflow (e.g.,bacterial, yeast, viral, etc.) or the volumes of materials beingprocessed. For example, while flat filters are relatively low cost andcommonly used, filters with a greater surface area, such as cylindricalfilters, may accept higher flow rates. In another example, hollowfilters may demonstrate lower recovery rates when processing smallvolumes of sample (e.g., less than about 10 ml). For example, for usewith bacteria, it may be preferable that the filter used is a membranefilter, particularly a hollow fiber filter. With the term “hollow fiber”is meant a tubular membrane. The internal diameter of the tube, in someexamples, is at least 0.1 mm, more preferably at least 0.5 mm, mostpreferably at least 0.75 mm and preferably the internal diameter of thetube is at most 10 mm, more preferably at most 6 mm, most preferably atmost 1 mm. Filter modules having hollow fibers are commerciallyavailable from various companies, including G.E. Life Sciences(Marlborough, Mass.) and InnovaPrep (Drexel, Mo.) (see, e.g.,US20110061474A1 to Page et al., entitled “Liquid to Liquid BiologicalParticle Concentrator with Disposable Fluid Path”).

In some implementations, the filtration module 1200 includes a filterejection means 1228 (e.g., actuator) to eject a filter 1230 post use.For example, a user or the robotic handling system may push the filter1230 into position for use such that the filter is retained by thefilter manifold 1220 during filtration. After filtration to remove theused filter 1230, the filter ejection actuator 1228 may eject the filter1230, releasing the filter 1230 such that the user or the robotichandling system may remove the used filter 1230 from the filtrationmodule 1200. The used filter 1230, in some examples, may be disposedwithin the solid waste module 1718 of FIGS. 17A and 17B, or returned toa filter cartridge 1240, as illustrated in FIG. 12D.

Turning to FIG. 12D, in some implementations, filters 1230 a, b, c, dprovided in the filter cartridge 1240 disposed within the chassis of theautomated instrument are transported to the filtration module 1200 by arobotic handling system (e.g., the robotic handling system 1708 of FIGS.17A and 17B) and positioned within the filtration module 1200 prior touse.

The filtration module 1200, in some implementations, requires periodiccleaning. For example, the processing system may alert a user whencleaning is required through the user interface of the automatedinstrument and/or through a wireless messaging means (e.g., textmessage, email, and/or personal computing device application). Adecontamination filter, for example, may be loaded into the filtrationmodule 1200 and the filtration module 1200 may be cleaned with a washsolution and/or alcohol mixture.

In some implementations, the filtration module 1200 is in fluidconnection with a wash cartridge 1210 (such as the wash cartridge 1100of FIG. 11A) containing the wash fluid via the connector 1218. Forexample, upon positioning by the user of the wash cartridge 1210 withinthe chassis of the automated instrument, the connector 1218 may matewith a bottom inlet of the wash cartridge 1210, creating a liquidpassage between the wash fluid 1206 and the filtration module 1200.

Turning to FIGS. 12B and 12C, in some implementations, a dual filterfiltration module 1250 includes dual filters 1252 a and 1252 b disposedover dual wash reservoirs 1254 a and 1254 b. In an example, each filtermay be a hollow core fiber filter having 0.45 um pores and greater than85 cm2 area. The wash reservoirs 1254 a and 1254 b, in some examples,may be 50 mL, 100 mL, or over 200 mL in volume. In some embodiments, thewash reservoirs 1254 a and 1254 b are disposed in a wash cartridge 1256,such as the wash or reagent cartridge 1100 of FIG. 11A.

The processing system of the automated instrument, in someimplementations, controls actuation of the dual filters 1252 a and 1252b in an X (horizontal) and Z (vertical) direction to position thefilters 1252 a, 1252 b in the wash reservoirs 1254 a and 1254 b. In aparticular example, the dual filters 1252 a and 1252 b may be move inconcert along the X axis but have independent Z axis range of motion.

As illustrated, the dual filters 1252 a and 1252 b of the filtrationmodule 1250 are connected to a slender arm body 1258. In someembodiments, any pumps and valves of the filtration module 1250 may bedisposed remotely from the body 1258 (e.g., within a floor of thechassis of the automated instrument). In this manner, the filtrationmodule 1250 may replace much bulkier conventional commercial filtrationmodules.

Further, in some embodiments, the filtration module 1250 is in liquidcommunication with a waste purge system designed to release liquid wasteinto a liquid waste storage unit, such as the liquid waste vessel 1208of FIG. 12A or the liquid waste storage module 1728 of FIGS. 17A and17B.

Wash and Reagent Cartridges

In some embodiments, the automated multi-module cell processinginstrument comprises one or more wash or reagent cartridges such asthose illustrated in FIGS. 11A-11E. The cartridge 1100 includes a pairof containers 1102 a, b, a set of four small tubes 1104 a, b, c, d, anda larger tube 1106 held in a cartridge body 1108. One or more of thecontainers 1102 a, b, and tubes 1104 a, b, c, d and 1106, in someembodiments, is sealed with a pierceable foil for access by an automatedliquid handling system, such as a sipper or pipettor. In otherembodiments, one or more of the containers 1102 a, b, and tubes 1104 a,b, c, d, and 1106 includes a sealable access gasket. The top of one ormore of the containers 1102 a, b, and tubes 1104 a, b, c, d, and 1106,in some embodiments, is marked with machine-readable indicia (notillustrated) for automated identification of the contents.

In some embodiments, containers 1102 a, b contain wash solutions. Thewash solution may be a same or different wash solutions. In someexamples, wash solutions may contain, e.g., buffer, buffer and 10%glycerol, 80% ethanol.

In some implementations, a cover 1110 secures the containers 1102 a, band tubes 1104 a, b, c, d and 1106 within the cartridge body 1108.Turning to FIG. 11B, the cover 1120 may include apertures for access toeach of the containers 1102 a, b and tubes 1104 a, b, c, d and 1106.Further, the cover 1120 may include machine-readable indicia 1112 foridentifying the type of cartridge (e.g., accessing a map of thecartridge contents). Alternatively, apertures may be marked separatelywith the individual contents.

In some embodiments, the reagent cartridge is a reagent cartridge suchas that illustrated in FIG. 11C. FIG. 11C shows a reagent cartridge 1126having a body 1122 including a set of eighteen tubes or vials 1128;however, the embodiment shown in FIG. 11C does not include an FTEPdevice. Looking at FIG. 11E, reagent cartridge includes sixteen tubes orvials 1126 a-p and an FTEP device 1124, held in a cartridge body 1122.One or more of the tubes or vials 1128 (FIG. 11C) or 1126 a-p (FIG.11E), in some embodiments, is sealed with pierceable foil for access byan automated liquid handling system, such as a sipper or pipettor. Inother embodiments such as that shown in FIG. 11E, one or more of thetubes or vials 1126 a-1126 p includes a sealable access gasket. The topof each of the small tubes or vials 1126 a-1126 p, in some embodiments,is marked with machine-readable indicia (not illustrated) for automatedidentification of the contents. The machine-readable indicia may includea bar code, QR code, or other machine-readable coding. Other automatedmeans for identifying a particular container can include color coding,symbol recognition (e.g., text, image, icon, etc.), and/or shaperecognition (e.g., a relative shape of the container). Rather than beingmarked upon the vessel itself, in some embodiments, an upper surface ofthe cartridge body and/or the cartridge cover may containmachine-readable indicia for identifying contents. The small tubes orvials may each be of a same size. Alternatively, multiple volumes oftubes or vials may be provided in the reagent cartridge 1100. In anillustrative example, each tube or vial may be designed to hold between2 and 20 mL, between 4 and 10 mL, or about 5 mL. In some embodimentswhere only small volumes of some reagents are required, tube inserts maybe used to accommodate small (e.g., microfuge) tubes in a largerreceptacle.

In an illustrative example, the tubes or vials 1126 a-1126 p may eachhold one the following materials: a vector backbone, oligonucleotides,reagents for nucleic acid assembly, a user-supplied cell sample, aninducer agent, magnetic beads in buffer, ethanol, an antibiotic for cellselection, reagents for eluting cells and nucleic acids, an oil overlay,other reagents, and cell growth and/or recovery media.

In some implementations, a cover 1120 as seen in FIG. 11D secures thetubes or vials 1128 within the cartridge body 1122 of FIG. 11C. Turningto FIG. 11D, the cover 1120 may include apertures for access to each ofthe small tubes or vials 1126. Three large apertures 1132 a-c areoutlined in a bold band to indicate positions to add user-suppliedmaterials. The user-supplied materials, for example, may include avector backbone, oligonucleotides, and a cell sample. Further, the cover1120 may include machine-readable indicia 1130 for identifying the typeof cartridge (e.g., accessing a map of the cartridge contents).Alternatively, each aperture may be marked separately with theindividual contents. In some implementations, to ensure positioning ofuser-supplied materials, the vials or tubes provided for filling in thelab environment may have unique shapes or sizes such that the cellsample vial or tube only fits in the cell sample aperture, theoligonucleotides vial or tube only fits in the oligonucleotidesaperture, and so on.

FTEP Modules

The FTEP (transformation) module may implement any cell transformationor transfection techniques routinely performed by electroporation.Electroporation is a widely-used method for permeabilization of cellmembranes that works by temporarily generating pores in the cellmembranes with electrical stimulation. The applications ofelectroporation include the delivery of DNA, RNA, siRNA, peptides,proteins, antibodies, drugs or other substances to a variety of cellssuch as mammalian cells (including human cells), plant cells, archea,yeasts, other eukaryotic cells, bacteria, and other cell types.Electrical stimulation may also be used for cell fusion in theproduction of hybridomas or other fused cells. During a typicalelectroporation procedure, cells are suspended in a buffer or mediumthat is favorable for cell survival. For bacterial cell electroporation,low conductance mediums, such as water, glycerol solutions and the like,are often used to reduce the heat production by transient high current.The cells and material to be electroporated into the cells (collectively“the cell sample”) is then placed in a cuvette embedded with two flatelectrodes for an electrical discharge. For example, Bio-Rad (Hercules,Calif.) makes the Gene Pulser Xcell™ line of products to electroporatecells in cuvettes. Traditionally, electroporation requires high fieldstrength.

Generally speaking, microfluidic electroporation—using cell suspensionvolumes of less than approximately 20 ml and as low as 1 μl—allows moreprecise control over a transfection or transformation process andpermits flexible integration with other cell processing tools comparedto bench-scale electroporation devices. Microfluidic electroporationthus provides unique advantages for, e.g., single cell transformation,processing and analysis; multi-unit FTEP device configurations; andintegrated, automated multi-module cell processing.

The present disclosure provides electroporation devices, modules, andmethods that achieve high efficiency cell electroporation with lowtoxicity where the electroporation devices and systems can be integratedwith other automated cell processing tools. Further, the electroporationdevice of the disclosure allows for multiplexing where two to manyelectroporation units are constructed and used in parallel, and allowsfor particularly easy integration with robotic liquid handlinginstrumentation. Such automated instrumentation includes, but is notlimited to, off-the-shelf automated liquid handling systems from Tecan(Mannedorf, Switzerland), Hamilton (Reno, Nev.), Beckman Coulter (FortCollins, Colo.), etc.

Although the disclosure is focused primarily on the above-described FTEPmodules for use in the instrument, a person of ordinary skill in the artwould understand upon reading the present disclosure that otherelectroporation and/or microfluidic devices could be used in theinvention in an equivalent fashion to that described herein. Specificelectroporation and microfluidic methods that can be used in the devicesof the disclosure include, but are not limited to, those described inU.S. App. No. 62/566,374; U.S. App. No. 62/566,375; U.S. App. No.62/551,069; U.S. App. No. 2017/0218355; WO 2017/040995; U.S. App. No.20150368604; U.S. App. No. 20070218355; U.S. App. No. 20110009807; U.S.Pat. Nos. 9,063,136; 9,029,109; 8,058,056; 7,951,582; 7,771,984;7,186,559; 6,492,175; Zhu T et al., Biomed Microdevices 12:35-40 (2010),T. Geng et al. Journal of Controlled Release 144: 91-100 (2010)h; Zhan,Y et al., (2011). Birck and NCN Publications. Paper 979; Adamo A et al.,Anal Chem. February 5; 85(3): 1637-1641 (2013); del Rosal et al., LabChip. October 7, 13(19): 3803-3821 (2013); Genga T and Lu C, Lab Chip,13, 3803-3821 (2013), Li, Y. et al., Sci. Rep. 5, 17817 (2015), Zhao, D.et al., Sci. Rep. 6, 18469 (2016), Garcia P. A. et al., Lab Chip, 17,490 (2017).

During the electroporation process, it is important to use voltagesufficient for achieving electroporation of material into the cells, butnot too much voltage as too much voltage will decrease cell viability.For example, to electroporate a suspension of a human cell line, 200volts is needed for a 0.2 ml sample in a 4 mm-gap cuvette withexponential discharge from a capacitor of about 1000 μF. However, if thesame 0.2 ml cell suspension is placed in a longer container with 2 cmelectrode distance (5 times of cuvette gap distance), the voltagerequired would be 1000 volts, but a capacitor of only 40 μF ( 1/25 of1000 μF) is needed because the electric energy from a capacitor followsthe equation of:E=0.5U²Cwhere E is electric energy, U is voltage and C is capacitance. Thereforea high voltage pulse generator is actually easy to manufacture becauseit needs a much smaller capacitor to store a similar amount of energy.Similarly, it would not be difficult to generate other wave forms ofhigher voltages.

The electroporation devices of the disclosure can allow for a high rateof cell transformation in a relatively short amount of time. The rate ofcell transformation is dependent on the cell type and the number ofcells being transformed. For example, for E. coli, the electroporationdevices can provide a cell transformation rate of 10³ to 10¹² cells persecond, 10⁴ to 10¹⁰ per second, 10⁵ to 10⁹ per second, or 10⁶ to 10⁸ persecond. Typically, 10⁸ yeast cells may be transformed per minute, and10¹⁰-10¹² bacterial cells may be transformed per minute. Theelectroporation devices also allow transformation of batches of cellsranging from 1 cell to 10¹² cells in a single transformation procedureusing parallel devices.

One embodiment of the FTEP device described herein is illustrated inFIGS. 4A-4C. FIG. 4A shows a planar top view of an FTEP device 400having an inlet 402 for introducing a fluid containing cells and nucleicacid to be delivered to the cells into the FTEP device 400 and an outlet404 for removing the transformed cells following electroporation. Ovalelectrodes 408 are positioned so as to define a center portion of theflow channel (not shown) where the channel narrows based on thecurvature of the electrodes. FIG. 4B shows a cutaway view from the topof the device 400, with the inlet 402, outlet 404, and electrodes 408positioned with respect to a flow channel 406. Note that the electrodes408 define a narrowing of flow channel 406. FIG. 4C shows a side cutawayview of the device 400 with the inlet 402 and inlet channel 412, andoutlet 404 and outlet channel 414. The electrodes 408 are oval in shapeand positioned so that they define a narrowed portion of the flowchannel 406.

In the FTEP devices of the disclosure, the toxicity level of thetransformation results in greater than 30% viable cells afterelectroporation, preferably greater than 35%, 40%, 45%, 50%, 55%, 60%,70%, 75%, 80%, 85%, 90%, 95% or even 99% viable cells followingtransformation, depending on the cell type and the nucleic acids beingintroduced into the cells.

The housing of the FTEP device can be made from many materials dependingon whether the FTEP device is to be reused, autoclaved, or isdisposable, including stainless steel, silicon, glass, resin, polyvinylchloride, polyethylene, polyamide, polystyrene, polyethylene,polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Similarly, the walls of the channels in thedevice can be made of any suitable material including silicone, resin,glass, glass fiber, polyvinyl chloride, polyethylene, polyamide,polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Preferred materials include crystal styrene,cyclo-olefin polymer (COP) and cyclic olephin co-polymers (COC), whichallow the device to be formed entirely by injection molding in one piecewith the exception of the electrodes and, e.g., a bottom sealing film ifpresent.

The FTEP devices described herein (or portions of the FTEP devices) canbe created or fabricated via various techniques, e.g., as entire devicesor by creation of structural layers that are fused or otherwise coupled.For example, for metal FTEP devices, fabrication may include precisionmechanical machining or laser machining; for silicon FTEP devices,fabrication may include dry or wet etching; for glass FTEP devices,fabrication may include dry or wet etching, powderblasting,sandblasting, or photostructuring; and for plastic FTEP devicesfabrication may include thermoforming, injection molding, hot embossing,or laser machining. The components of the FTEP devices may bemanufactured separately and then assembled, or certain components of theFTEP devices (or even the entire FTEP device except for the electrodes)may be manufactured (e.g., using 3D printing) or molded (e.g., usinginjection molding) as a single entity, with other components added aftermolding. For example, housing and channels may be manufactured or moldedas a single entity, with the electrodes (and, e.g., bottom sealing film)later added to form the FTEP module (see, FIG. 10F (i)). Alternatively,the FTEP device may also be formed in two or more parallel layers, e.g.,a layer with the horizontal channel and filter, a layer with thevertical channels, and a layer with the inlet and outlet ports, whichare manufactured and/or molded individually and assembled followingmanufacture. (See, e.g., FIG. 9A.)

In specific aspects, the FTEP device can be manufactured using a circuitboard as a base, with the electrodes, filter and/or the flow channelformed in the desired configuration on the circuit board, and theremaining housing of the device containing, e.g., the one or more inletand outlet channels and/or the flow channel formed as a separate layerthat is then sealed onto the circuit board. The sealing of the top ofthe housing onto the circuit board provides the desired configuration ofthe different elements of the FTEP devices of the disclosure. Also, twoto many FTEP devices may be manufactured on a single substrate, thenseparated from one another thereafter or used in parallel. In certainembodiments, the FTEP devices are reusable and, in some embodiments, theFTEP devices are disposable. In additional embodiments, the FTEP devicesmay be autoclavable.

The electrodes 408 can be formed from any suitable metal, such ascopper, stainless steel, titanium, aluminum, brass, silver, rhodium,gold or platinum, or graphite. One preferred electrode material is alloy303 (UNS330300) austenitic stainless steel. An applied electric fieldcan destroy electrodes made from of metals like aluminum. If amultiple-use (i.e., non-disposable) flow-through FTEP device isdesired—as opposed to a disposable, one-use flow-through FTEP device—theelectrode plates can be coated with metals resistant to electrochemicalcorrosion. Conductive coatings like noble metals, e.g., gold, can beused to protect the electrode plates.

Additionally, the FTEP devices may comprise push-pull pneumatic means toallow multi-pass electroporation procedures; that is, cells toelectroporated may be “pulled” from the inlet toward the outlet for onepass of electroporation, then be “pushed” from the outlet end of theflow-through FTEP device toward the inlet end to pass between theelectrodes again for another pass of electroporation. This process maybe repeated one to many times.

Depending on the type of cells to be electroporated (e.g., bacterial,yeast, mammalian) and the configuration of the electrodes, the distancebetween the electrodes in the flow channel can vary widely. For example,in the embodiments shown in FIGS. 4A-4I, 5A-5H, 6, and 7A-7E where theelectrodes form a portion of the wall of the flow channel where the flowchannel decreases in width, the distance between the electrodes in theflow channel may be between 10 μm and 5 mm, or between 25 μm and 3 mm,or between 50 μm and 2 mm, or between 75 μm and 1 mm. In otherembodiments such as those depicted in FIGS. 8A-8U, 9A-9C, and 10A-10Dwhere the electrodes are positioned on either end of the channelnarrowing, the distance between the electrodes in the flow channel maybe between 1 mm and 10 mm, or between 2 mm and 8 mm, or between 3 mm and7 mm, or between 4 mm and 6 mm. The overall size of the FTEP device maybe from 3 cm to 15 cm in length, or 4 cm to 12 cm in length, or 4.5 cmto 10 cm in length. The overall width of the FTEP device may be from 0.5cm to 5 cm, or from 0.75 cm to 3 cm, or from 1 cm to 2.5 cm, or from 1cm to 1.5 cm.

The region of the flow channel that is narrowed is typically wide enoughso that at least two cells can fit in the narrowed portion side-by-side.For example, a typical bacterial cell is 1 μm in diameter; thus, thenarrowed portion of the flow channel of the FTEP device used totransform such bacterial cells will be at least 2 μm wide. In anotherexample, if a mammalian cell is approximately 50 μm in diameter, thenarrowed portion of the flow channel of the FTEP device used totransform such mammalian cells will be at least 100 μm wide. That is,the narrowed portion of the FTEP device will not physically contort or“squeeze” the cells being transformed.

In embodiments of the FTEP device where reservoirs are used to introducecells and nucleic acid into the FTEP device, the reservoirs range involume from 100 μL to 10 mL, or from 500 μL to 75 mL, or from 1 mL to 5mL. The flow rate in the FTEP ranges from 0.1 mL to 5 mL per minute, orfrom 0.5 mL to 3 mL per minute, or from 1.0 mL to 2.5 mL per minute. Thepressure in the FTEP device ranges from 1-30 psi, or from 2-10 psi, orfrom 3-5 psi.

To avoid different field intensities between the electrodes, theelectrodes should be arranged in parallel. Furthermore, the surface ofthe electrodes should be as smooth as possible without pin holes orpeaks. Electrodes having a roughness Rz of 1 to 10 μm are preferred. Inanother embodiment of the invention, the flow-through electroporationdevice comprises at least one additional electrode which applies aground potential to the FTEP device.

The electrodes are configured to deliver 1-25 Kv/cm, or 5-20 Kv/cm, or10-20 Kv/cm. The further apart the electrodes are, the more voltageneeds to be supplied; in addition, the voltage delivered of coursedepends on the types of cells being porated, the medium in which thecells are suspended, the size of the electroporation channel, and thelength and diameter of the electrodes. There are many different pulseforms that may be employed with the FTEP device, including exponentialdecay waves, square or rectangular waves, arbitrary wave forms, or aselected combination of wave forms. One type of common pulse form is theexponential decay wave, typically made by discharging a loaded capacitorto the cell sample. The exponential decay wave can be made less steep bylinking an inductor to the cell sample so that the initial peak currentcan be attenuated. When multiple waveforms in a specified sequence areused, they can be in the same direction (direct current) or differentdirections (alternating current). Using alternating current can bebeneficial in that two topical surfaces of a cell instead of just onecan be used for molecular transport, and alternating current can preventelectrolysis. The pulse generator can be controlled by a digital oranalog panel. In some embodiments, square wave forms are preferred, andin other embodiments, an initial wave spike before the square wave ispreferred.

The FTEP device may be configured to electroporate cell sample volumesbetween 1 μl to 5 ml, 10 μl to 2 ml, 25 μl to 1 ml, or 50 μl to 750 μl.The medium or buffer used to suspend the cells and material (reagent) tobe electroporated into the cells for the electroporation process may beany suitable medium or buffer, such as MEM, DMEM, IMDM, RPMI, Hanks',PBS and Ringer's solution, where the media may be provided in a reagentcartridge as part of a kit. Further, because the cells must be madeelectrocompetent prior to transformation or transfection, the bufferalso may comprise glycerol or sorbitol, and may also comprise asurfactant. For electroporation of most eukaryotic cells the medium orbuffer usually contains salts to maintain a proper osmotic pressure. Thesalts in the medium or buffer also render the medium conductive. Forelectroporation of very small prokaryotic cells such as bacteria,sometimes water or 10% glycerol is used as a low conductance medium toallow a very high electric field strength. In that case, the chargedmolecules to be delivered still render water-based medium moreconductive than the lipid-based cell membranes and the medium may stillbe roughly considered as conductive particularly in comparison to cellmembranes.

The compound to be electroporated into the cells can be any compoundknown in the art to be useful for electroporation, such as nucleicacids, oligonucleotides, polynucleotides, DNA, RNA, peptides, proteinsand small molecules like hormones, cytokines, chemokines, drugs, or drugprecursors.

Another embodiment of the FTEP devices described herein is illustratedin FIGS. 4D-4F. FIG. 4D shows a top planar view of an FTEP device 410having an inlet 402 for introducing a fluid containing cells and nucleicacid into the FTEP device 410 and an outlet 404 for removing thetransformed cells following electroporation. Cylindrical electrodes 408are positioned so as to define a center portion of the flow channel (notshown) where the flow channel narrows as a result of the curvature ofthe electrodes. FIG. 4E shows a cutaway view from the top of the FTEPdevice 410, with the inlet 402, outlet 404, and electrodes 408positioned with respect to a flow channel 406. Again, note that theelectrodes 408 define a narrowed portion or region of flow channel 406.FIG. 4F shows a side cutaway view of FTEP device 410 with the inlet 402and inlet channel 412, and outlet 404 and outlet channel 414. Theelectrodes 408 are cylindrical and positioned in the flow channel 406defining a narrowed portion of the flow channel 406.

Yet another embodiment of the FTEP devices of the disclosure isillustrated in FIGS. 4G-4I. FIG. 4G shows a top planar view of an FTEPdevice 420 having an inlet 402 for introducing a fluid containing cellsand nucleic acid into FTEP device 420, and an outlet 404 for removingthe transformed cells following electroporation. The semi-cylindricalelectrodes 408 are positioned so as to define a narrowed portion of aflow channel (not shown) where the channel narrows from both ends basedon the curvature of the electrodes. FIG. 4H shows a cutaway view fromthe top of FTEP device 420, with the inlet 402, outlet 404, andelectrodes 408 positioned with respect to a flow channel 406. FIG. 4Ishows a side cutaway view of FTEP device 420 with inlet 402 and inletchannel 412, and outlet 404 and outlet channel 414. The semi-cylindricalelectrodes 408 are positioned in the flow channel 406 so that theydefine a narrowed portion of the flow channel 406. It should be notedthat the devices depicted in FIGS. 4A-4I show the electrodes positionedsubstantially mid-way along the flow channel; however, in other aspectsof the devices, the electrodes may be positioned in narrowed regions ofthe flow channel more toward the inlet of the FTEP device or more towardthe outlet of the FTEP device.

FIGS. 5A-5E show embodiments of the FTEP devices of the disclosure withseparate inlets for the cells and the nucleic acid. FIG. 5A shows a topplanar view of an FTEP device 500 having a first inlet 502 forintroducing a fluid containing cells into FTEP device 500; a secondinlet 518 for introducing a fluid containing nucleic acids to beelectroporated into the cells into FTEP device 500; electrodes 508; andan outlet 504 for removing the transformed cells followingelectroporation. Although these embodiments are illustrated withcylindrical electrodes, as shown in FIG. 5A, other shaped electrodeswith a curved edge—e.g., oval, semi-cylindrical, and the like as shownin relation to FIGS. 4A-4I—may be used to define the flow channel. FIG.5B shows a cutaway view from the top of FTEP device 500, with the firstinlet 502, second inlet 518, outlet 504, and electrodes 508 positionedwith respect to the flow channel 506.

FIG. 5C shows a cutaway view of the embodiment of FTEP device 500 withthe first inlet 502 and second inlet 518 positioned as shown in FIGS. 5Aand 5B. In FIG. 5C, the first inlet channel 512 and second inlet channel522 meet independently with flow channel 506, and the liquid (cells andmaterial to be porated or delivered to the cells) flows through the flowchannel 506 to the outlet channel 514 and outlet 504 where thetransformed cells are removed from the FTEP device. The electrodes 508are positioned in the flow channel 506 so that they define a narrowedportion of the flow channel 506. FIG. 5D shows a side cutaway view of avariation 510 on the embodiment of the FTEP device 500 depicted in FIGS.5A and 5B. Here, the first inlet channel 512 and second inlet channel524 intersect with the flow channel 506 at a three-way junction, and theliquid (cells and material to be porated or delivered to the cells)flows through the flow channel 506 to the outlet channel 514 and outlet504 where the transformed cells are removed from the FTEP device. Theelectrodes 508 are positioned in the flow channel 506 defining anarrowed portion of the flow channel 506. FIG. 5E shows a first sidecutaway view 520 of a yet another variation of the FTEP device 500 shownin FIGS. 5A and 5B. Here, the first inlet channel 512 and second inletchannel 526 intersect at a junction where the cells and nucleic acidsmix prior to introduction of the combined fluids to the flow channel506. The fluids flow through the flow channel 506 to the outlet channel514 and outlet 504 where the transformed cells are removed from the FTEPdevice. Electrodes 508 are positioned in the flow channel 506 so thatthey define a narrowed portion of the flow channel 506.

FIGS. 5F-5H show another embodiment of the FTEP devices of thedisclosure with separate inlets for the cells and the nucleic acid. FIG.5F shows a top planar view of an electroporation device 530 having afirst inlet 502 for introducing a fluid containing cells, a secondoutlet 518 for introducing nucleic acids to be electroporated into thecells, and an outlet 504 for removing the transformed cells followingelectroporation. The electrodes 508 are positioned between the firstinlet 502 where the cells are introduced into the FTEP device and thesecond inlet 518 where the nucleic acids are introduced into the FTEPdevice. FIG. 5G shows a cutaway view from the top of the FTEP device530, with the first inlet 502, second inlet 518, and outlet 504, andwith electrodes 508 positioned between the first inlet channel 502 andthe second inlet channel 518, where the electrodes 508 form a narrowedportion of flow channel 506. FIG. 5H shows a side cutaway view of FTEPdevice 530 with the first inlet 502 where the cells are introduced intothe FTEP device and first inlet channel 512, the second inlet 518 wherethe nucleic acids are introduced into the FTEP device and second inletchannel 532, and an outlet channel 514 and outlet 504 where thetransformed cells are removed from the FTEP device. The electrodes 508are positioned in the flow channel 506 defining a narrow portion of theflow channel 506 and are positioned between the first inlet channel 512and the second inlet channel 532 such that the material to be introducedinto the cells is added to the fluid comprising the cells after thecells have been electroporated.

FIG. 6 illustrates an FTEP device in which the flow of the fluidintroduced into the flow channel from the input channel(s) is focused,e.g., using an immiscible fluid such as an oil or a stream of air tonarrow the stream of the fluid containing the cells and the nucleicacids as it passes by the electrodes. FIG. 6 shows a cutaway view fromthe top of the FTEP device 600, with the inlet 602, outlet 604, and theelectrodes 608 positioned between the first inlet channel 602 and outlet604. The flow focusing 630 is effected by an immiscible fluid, where theelectrodes 608 form a narrowed portion of flow channel 606. (For methodsand inlet configurations relevant to flow focusing, see, e.g., US Pub.Nol. 2010/0184928 to Kumacheva.)

Multiplexed embodiments of exemplary FTEP devices are illustrated inFIGS. 7A-7E. FIG. 7A illustrates a top view of a cross section of afirst multiplexed aspect of the FTEP devices of the disclosure. The FTEPdevice in FIG. 7A is a multiplexed FTEP device 700 in which parallelflow channels 706 for each FTEP module are defined in part by sharedcylindrical electrodes 708 a-708 f forming devices (i), (ii), (iii),(iv), and (v). Each flow channel 706 has an inlet 702 for introducingdifferent sets of cells and/or nucleic acids into the FTEP units and anoutlet 704 for removing the transformed cells from the FTEP units.Adjacent units share electrodes, where the electrodes alternate charge,e.g., +/−/+/−/+ (that is, if electrode 708 a is +, electrode 708 b is −,electrode 708 c is +, electrode 708 d is −, and so on). FIG. 7B is anillustration of a top view of a cross section of a second multiplexedembodiment of the FTEP devices 710 of the disclosure. This is amultiplexed device 710 in which parallel flow channels 706 are definedin part by shared oval electrodes 708 a-708 f. Each flow channel 706 hasan inlet 702 for introducing different sets of cells and/or nucleicacids into the flow channels 706, and an outlet for removing thetransformed cells from FTEP units (i), (ii), (iii), (iv), and (v).Again, adjacent devices share electrodes, where the electrodes alternatecharge, e.g., +/−/+/−/+.

FIG. 7C is an illustration of a top view of a cross section of a thirdmultiplexed embodiment of the FTEP devices of the disclosure. In thisexemplary multiplexed FTEP device 720, the individual FTEP units arestaggered. The parallel flow channels 706 are defined in part byindividual cylindrical electrodes 708 a-708 j that are not shared asshown in FIGS. 7A and 7B. Each flow channel 706 has its own pair ofelectrodes 708, an inlet 702 for introducing different sets of cellsand/or nucleic acids into the FTEP device, and an outlet for removingtransformed cells from the FTEP units (i), (ii), (iii), (iv), and (v).FIG. 7D is an illustration of a top view of a cross section of anotherexemplary multiplexed FTEP device. In this multiplexed FTEP device 730,staggered, parallel flow channels 706 are defined in part by individualoval electrodes 708 a-708 j. Each flow channel 706 has its own un-sharedpair of electrodes 708 (e.g., 708 a/708 b, 708 c/708 d, 708 e/708 f, 708g/708 h, and 708 i/708 j), an inlet 702 for introducing different setsof cells and/or nucleic acids into the FTEP units, and an outlet 704 forremoving transformed cells from the FTEP units. FIG. 7E is anillustration of a top view of a cross section of another exemplarymultiplexed FTEP device. In this exemplary multiplexed device 740,staggered, parallel flow channels 706 are defined in part by individualhalf-cylindrical electrodes 708 a-708 j. Each flow channel 706 has itsown pair of electrodes 708, a separate inlet 702 for introducingdifferent sets of cells and/or nucleic acids into the FTEP unit, and anoutlet 704 for removing the transformed cells from the FTEP unit.

Additional embodiments of the FTEP devices of the disclosure areillustrated in FIGS. 8A-8U. Note that in the FTEP devices in FIGS. 8A-8Uthe electrodes are not positioned on either side of the flow channel tonarrow the flow channel; instead, the electrodes are placed such that afirst electrode is placed between the inlet and the narrowed region ofthe flow channel, and the second electrode is placed between thenarrowed region of the flow channel and the outlet. FIG. 8A shows a topplanar view of an FTEP device 800 having an inlet 802 for introducing afluid containing cells and nucleic acid into FTEP device 800 and anoutlet 804 for removing the transformed cells from the FTEP followingelectroporation. The electrodes 808 are introduced through channels (notshown) in the device. FIG. 8B shows a cutaway view from the top of theFTEP device 800, with the inlet 802, outlet 804, and electrodes 808positioned with respect to a flow channel 806. FIG. 8C shows a sidecutaway view of FTEP device 800 with the inlet 802 and inlet channel812, and outlet 804 and outlet channel 814. The electrodes 808 arepositioned in electrode channels 816 so that they are in fluidcommunication with the flow channel 806, but not directly in the path ofthe cells traveling through the flow channel 806. Again note that thefirst electrode is placed between the inlet and the narrowed region ofthe flow channel, and the second electrode is placed between thenarrowed region of the flow channel and the outlet.

An expanded side cutaway view of the bottom portion of the device 800 inFIG. 8D shows that the electrodes 808 in this aspect of the device arepositioned in the electrode channels 816 which are generallyperpendicular to the flow channel 806 such that the fluid containing thecells and nucleic acid flows from the inlet channel 812 through the flowchannel 806 to the outlet channel 814, and in the process fluid flowsinto the electrode channels 816 to be in contact with the electrodes808. In this aspect, the inlet channel, outlet channel and electrodechannels all originate from the same planar side of the device, as shownin FIGS. 8C and 8D. In certain aspects, however, such as that shown inFIG. 8E, the electrodes are introduced from a different planar side ofthe FTEP device than the inlet and outlet channels. Here, the electrodes808 in this alternative aspect of FTEP device 810 are positioned in theelectrode channels 816 perpendicular to the flow channel 806 such thatfluid containing the cells and nucleic acid flow from the inlet channel812 through the flow channel 806 to the outlet channel 814. The cellsand nucleic acid in buffer flow into the electrode channels 816 to be incontact with both electrodes 808; however, the electrodes 808 are notdirectly in flow channel 806. In this aspect, the inlet channel andoutlet channel originate from a different planar side of the device thando the electrodes and electrode channels.

FIGS. 8F-8H illustrate yet another aspect of the FTEP devices of thedisclosure. FIG. 8F shows a top planar view of an FTEP device 820 havinga first inlet 802 for introducing a fluid containing cells into FTEPdevice 820 and an outlet 804 for removing the transformed cells from theFTEP device 820 following electroporation. However, in this FTEP device,there is a second inlet 822 for introducing nucleic acid to beelectroporated to the cells. The electrodes 808 are introduced throughchannels (not shown). FIG. 8G shows a cutaway view from the top of theFTEP device 820, with the first inlet 802, second inlet 822, outlet 804,and the electrodes 808 positioned with respect to the flow channel 806.FIG. 8H shows a side cutaway view of FTEP device 820 with inlets 802,822 and inlet channels 812, 824 and outlet 804 and outlet channel 814.The electrodes 808 are positioned in the electrode channels 816 so thatthey are in fluid communication with the flow channel 806, but notsubstantially in the path of the cells traveling through the flowchannel 806. The electrodes 808 in this aspect of the FTEP device 820are positioned in the electrode channels 816 where the electrodechannels 816 are generally perpendicular to the flow channel 806 suchthat fluid containing the cells and fluid containing the nucleic acidsflow from the inlets 802, 822 through the inlet channels 812, 824 intothe flow channel 806 and through to the outlet channel 814, and in theprocess the cells and nucleic acid in medium flows into the electrodechannels 816 to be in contact with the electrodes 808. One of the twoelectrodes 808 and electrode channels 816 is positioned between inlets802 and 822 and inlet channels 812 and 824 and the narrowed region (notshown) of flow channel 806, and the other electrode 808 and electrodechannel 816 is positioned between the narrowed region (not shown) offlow channel 806 and the outlet channel 814 and outlet 804. In FIG. 8H,the inlet channel, outlet channel and electrode channels all originatefrom the same planar side of the device, although the electrodes (andinlets and outlet) can also be configured to originate from a differentplanar sides of the FTEP device such as illustrated in FIG. 8E.

FIGS. 8I-8M illustrate yet another embodiment of the devices of thedisclosure. FIG. 8I shows a top planar view of an electroporation device830 having an inlet 802 for introducing a fluid containing cells andnucleic acid into the FTEP device 830 and an outlet 804 for removal ofthe transformed cells from the FTEP device 8300 followingelectroporation. The electrodes 808 are introduced through channels (notshown) machined into the device. FIG. 8J shows a cutaway view from thetop of the device 830, showing an inlet 802, an outlet 804, a filter 850of substantially uniform density, and electrodes 808 positioned withrespect to the flow channel 806. FIG. 8K shows a cutaway view from thetop of an alternative configuration 840 of the device 830, with an inlet802, an outlet 804, a filter 850 of increasing gradient density, andelectrodes 808 positioned with respect to the flow channel 806. In FIGS.8I-8M, like FIGS. 8F-8H, the first electrode is placed between the inletand the narrowed region of the flow channel, and the second electrode isplaced between the narrowed region of the flow channel and the outlet.In some embodiments such as those depicted in FIGS. 8I-8M, the FTEPdevices comprise a filter disposed within the flow channel positioned inthe flow channel after the inlet channel and before the first electrodechannel. The filter may be substantially homogeneous in porosity (e.g.,have a uniform density as in FIG. 8J); alternatively, the filter mayincrease in gradient density where the end of the filter proximal to theinlet is less dense, and the end of the filter proximal to the outlet ismore dense (as shown in FIG. 8K). The filter may be fashioned from anysuitable and preferably inexpensive material, including porous plastics,hydrophobic polyethylene, cotton, glass fibers, or the filter may beintegral with and fabricated as part of the FTEP device body (see, e.g.,FIG. 10E).

FIG. 8L shows a side cutaway view of the device 840 with an inlet 802and an inlet channel 812, and an outlet 804 and an outlet channel 814.The electrodes 808 are positioned in the electrode channels 816 so thatthey are in fluid communication with the flow channel 806, but notdirectly in the path of the cells traveling through flow channel 806.Note that filter 850 is positioned between inlet 802 and inlet channel812 and electrodes 808 and electrode channels 816. An expanded sidecutaway view of the bottom portion of the FTEP device 840 in FIG. 8Mshows that the electrodes 808 in this aspect of the FTEP device 840 arepositioned in the electrode channels 816 and perpendicular to the flowchannel 806 such that fluid containing the cells and nucleic acid flowsfrom the inlet channel 812 through the flow channel 806 to the outletchannel 814, and in the process fluid flows into the electrode channels816 to be in contact with both electrodes 808. In FIGS. 8L and 8M, theinlet channel, outlet channel and electrode channels all originate fromthe same planar side of the device, although the electrodes (and theinlets and outlet) can also be configured to originate from a differentplanar side such as illustrated in FIG. 8E.

FIGS. 8N-8R illustrate other embodiments of the FTEP devices of thedisclosure. FIG. 8N shows a top view of an FTEP device 860 having afirst inlet 802 for introducing a fluid containing cells into the FTEPdevice and a second inlet 818 for introducing a fluid containing nucleicacids to be introduced to the cells into the FTEP device, electrodes 808positioned in electrode channels (not shown), and an outlet 804 forremoval of the transformed cells following electroporation. FIG. 8Oshows a cutaway view from the top of the device 860, comprising a firstinlet 802, second inlet 818, outlet 804, filter 850, and electrodes 808positioned with respect to the flow channel 806. Again note that theelectrodes 808 are positioned so that the first electrode is on the“inlet end” of the narrowed region in flow channel 806 and the secondelectrode is on the “outlet end” of the narrowed region in flow channel806. FIG. 8P shows a first side cutaway view of an embodiment of thedevice 860 with the first inlet 802 and second inlet 818 positioned asshown in FIG. 8N. The first inlet channel 812 and second inlet channel824 meet separately with the flow channel 806 prior to encounteringfilter 850, and the liquid flows from the inlet channels 812 and 824through the flow channel 806 (and filter 850) to the outlet channel 814and outlet 804. Note that in some embodiments, electrodes 808 may bepositioned in electrode channels 816 such that electrodes 808 are flushwith the walls of flow channel 806 (e.g., see FIG. 10F(iii)).Alternatively, electrodes 808 may extend a minimal distance into flowchannel 806; however, in doing so electrodes 808 do not extend into flowchannel 806 to the extent that the electrodes impede the flow of thecells through the flow channel.

FIG. 8Q shows a side cutaway view of a variation of the embodiment ofthe FTEP device 860 shown in FIGS. 8N-8P with the first inlet 802 andsecond inlet 818 positioned as shown in FIG. 8N. The first inlet channel812 and second inlet channel 824 intersect with flow channel 806 at athree-way junction with flow channel 806 and prior to encounteringfilter 850. The liquid flows through the flow channel 806 to the outletchannel 824 and outlet 804. The electrodes 808 are positioned in theelectrode channels 816 so that they are in fluid communication with theflow channel 806, but not directly in the path of the cells travelingthrough the flow channel 806. Again, the electrodes 808 are positionedso that the first electrode is on the “inlet end” of the narrowed regionin flow channel 806 and the second electrode is on the “outlet end” ofthe narrowed region in flow channel 806. FIG. 8R shows a side cutawayview of yet another variation on the embodiment of the FTEP device 860shown in FIGS. 8N-8P. The first inlet channel 812 and second inletchannel 826 intersect at a junction into a single channel prior tointersecting flow channel 806. The fluids flow from the inlets 802 and818, through the inlet channels 812 and 826, into and through flowchannel 806 and the filter 850, into electrode channels 816 (such thatelectrodes 808 are in fluid communication with flow channel 806) andcontinuing through flow channel 806 to the outlet channel 814 andfinally to the outlet 804 where the transformed cells are removed fromthe FTEP device 860. Again in this embodiment, the electrodes 808 arepositioned in the electrode channels 816 so that they are in fluidcommunication with the flow channel 806, but not directly in the flowpath of the cells traveling through the flow channel 806. Although eachof FIGS. 8P-8R show the inlet channels, outlet channel and electrodechannels originating from the same planar side of the device, all of theinlets, outlet and electrodes in each of these aspects can also beconfigured to originate from different planar sides of the FTEP device.

FIGS. 8S-8U illustrate another embodiment of the FTEP devices of thedisclosure. FIG. 8S shows a top view of an electroporation device 870having a first inlet 802 for introducing a fluid containing cells intoFTEP device 870, a second inlet 818 for introducing nucleic acids to beporated into the cells into FTEP device 870, and an outlet 804 forremoving transformed cells from FTEP device 870 followingelectroporation. The electrodes 808 are introduced through channels (notshown) machined into the device and are positioned between the firstinlet 802 and the second inlet 818. FIG. 8T shows a cutaway view fromthe top of the device 870, with the first inlet 802, second inlet 818,outlet 804, and the electrodes 808 positioned with respect to the flowchannel 806. Additionally, the FTEP device depicted in FIG. 8T comprisesa filter 850 disposed between the first inlet 802 and the firstelectrode 808 and before the narrowed region of flow channel 806. Filter850 in this embodiment has a gradient of pore sizes, from large to small(moving from the inlet 802 toward the narrowed portion of flow channel806. FIG. 8U shows a side cutaway view of FTEP device 870 comprising afirst inlet 802 and first inlet channel 812, a filter 850, a secondinlet 818 and second inlet channel 832, and an outlet 804 and outletchannel 814. The electrodes 808 are positioned in the electrode channels816 perpendicular to flow channel 806 and between the first and secondinlets. The electrodes 808 are in fluid communication with flow channel806, but not in the flow channel and thus in the path of the cellstraveling through flow channel 806. Nucleic acids are added to FTEPdevice 870 via the second inlet 818 and through the second inlet channel832 and encounter the cells after the cells are electroporated. In FIG.8U, the inlet channels, outlet channel and electrode channels alloriginate from the same planar side of the device, although thesefeatures can also be configured to originate from different planar sidesof FTEP device 870.

FIGS. 9A and 9B show the side and top cutaway views, respectively, ofyet another embodiment of the invention. FIG. 9A shows a multilayerdevice 900 with a top layer 952 having an inlet 902 and an inlet channel912, a flow channel 906, and outlet 904 and an outlet channel 914. Theelectrodes 908 are on bottom layer 956, e.g., provided as strips on asolid substrate. The middle layer 954 is a solid substrate withelectrode channels 916 provided therein, and the electrode channels 916in this aspect provide fluid communication between the electrodes 908 ofbottom layer 956 and flow channel 906 of top layer 952. The cells andnucleic acids in fluid are introduced to the FTEP device 900 via inlet902 and flow through inlet channel 912 and into flow channel 906, andthen to the outlet channel 914. In the process, the fluid flows intoelectrode channels 916 so that electrodes 908 are in fluid contact withflow channel 906. The cells are porated as they pass through flowchannel 906 between the two electrodes 908. FIG. 9B shows the top viewof a cutaway 910 of the embodiment of the FTEP device 900 showing theposition of the inlet 902, outlet 904, electrodes 908 and electrodechannels 916 with respect to the flow channel 906. Although theelectrodes are shown here as strips, they may also be configured to beother shapes, e.g., round, cylindrical, asymmetric, rectangular, orsquare.

FIG. 9C illustrates an FTEP device in which flow focusing 930 of thefluid introduced into the flow channel from the input channel(s) takesplace, e.g., using an immiscible fluid such as an oil or using air tofocus (narrow) the stream of the fluid containing the cells and nucleicacids as the fluid encounters the electrode channels, and theelectrodes. FIG. 9C shows a cutaway view from the top of the device 920,with the first inlet 902, the flow focusing 930 of the fluid after itexits the inlet channel and enters the flow channel 906, and theelectrodes 908 positioned between the inlet 902 and the outlet 904,where the electrodes 908 are positioned on either end of a narrowedportion of flow channel 906.

The reagent cartridges for use in the automated instruments (e.g.,cartridge 1122 of FIG. 11E), in some embodiments, include one or moreFTEP devices (e.g., electroporation module 1124 of FIG. 11E). FIGS. 10Athrough 10C are top perspective, bottom perspective, and bottom views,respectively, of six co-joined FTEP devices 1050 that may be part of,e.g., reagent cartridge 1122 in FIG. 11E infra (i.e., serve as FTEP 1124in reagent cartridge 1122). FIG. 10A depicts six FTEP units 1050 (i.e.,(i), (ii), (iii), (iv), (v), and (vi)) arranged on a single,integrally-formed injection molded cyclic olefin copolymer (COC)substrate 1056. The channels 1006 shown in FIG. 10B are sealed with aCOC film having a thickness of 50 microns to 1 mm (not shown). The COCfilm may be thermally bonded to the base of the assembly 1000 (thesurface most prominently displayed in FIG. 10B). In FIGS. 10B and 10C,the co-joined FTEP devices have different channel architectures andelectrode placements that may be advantageous in various applications.For instance, the curved channels of devices (i), (iv) and (v) takeadvantage of inertia to direct the cells in the fluid away from theelectrodes. The electrodes may be positioned off center in the channelto further enhance cells flow and reduce the potential for damage to thecells. This may be particularly important for cells or materials thatare particularly sensitive to electrolytic effects or local changes inpH proximate the electrodes. The electrodes may be at least partiallyembedded into the channel walls, as shown in embodiments (iii) and (iv),so as to further reduce these effects.

Each of the six FTEP units 1050 have wells or reservoirs 1052 thatdefine cell sample inlets and wells 1054 that define cell sampleoutlets. FIG. 10B is a bottom perspective view of the six co-joined FTEPdevices 1050 of FIG. 15A also depicting six FTEP units 1050 (i.e.,(i)-(vi)) arranged on a single substrate 1056. Six inlet wells 1052 canbe seen, one for each flow-through electroporation unit 1050, and oneoutlet well 1054 can be seen. Also seen in FIG. 10B for each FTEP unit1050 are an inlet 1002, an outlet 1004, a flow channel 1006, and twoelectrodes 1008 on either end of a narrowed region in flow channel 1006.Filters 1070 and 1072 are included in the channels to prevent cloggingof the channel, particularly at narrowed region of the flow channel.FIG. 10C is a bottom view of the six co-joined FTEP devices 1050 ofFIGS. 10A and 10B. Depicted in FIG. 10C are six FTEP units 1050 (i.e.,(i)-(vi)) arranged on a single substrate 1056, where each FTEP unit 1050comprises an inlet 1002, outlet 1004, flow channel 1006 and twoelectrodes 1508 on either end of a narrowed region in flow channel 1006in each FTEP unit 1050. Once the six FTEP units 1050 are fabricated,they can be separated from one another (e.g., “snapped apart”) upon thedepicted score lines and used one at a time as seen in the cartridgedepicted in FIG. 11E; alternatively, the FTEP units may be used inembodiments where two or more FTEP units 1050 are used in parallel.

FIG. 10D shows scanning electromicrographs of the FTEP units depicted inFIG. 10C with the units (i), (ii), (iii), (iv), (v), and (vi) in FIG.10D corresponding to units (i), (ii), (iii), (iv), (v), and (vi) in FIG.10C. In FIG. 10D, for each unit both the electrode channels 1016 as wellas the flow channel 1006 can be seen.

FIG. 10E shows scanning electromicrographs of the filters 1070 and 1072depicted as black bars in FIGS. 10B and 10C. Note in this embodiment,the porosity of the filter 1072 varies from large pores (near the inlet1002) to small pores toward the flow channel (not shown). In thisembodiment, the channel optionally but not necessarily narrows. If asecond filter is present, the second filter may also vary in porosity.In the case of a second filter between the second electrode and theoutlet channel, the filter can vary from large pores (near the secondelectrode) to small pores toward the outlet channel. Scale informationis shown in each micrograph.

In certain embodiments, the filter serves the purpose of filtering thefluid containing the cells and DNA before the fluid encounters thenarrowed portion of the flow channel. The filter thus decreases thelikelihood that cells or other matter do not clog the narrowed portionof the flow channel. Instead, if there is particulate matter that posesa threat to clogging the narrowed portion of the flow channel, thefilter will catch the particulate matter leaving other pores throughwhich the rest of the cell/DNA/fluid can move. The depicted construction(integral molding with the channel wall) is particularly advantageousbecause it reduces cost and complexity of the device while also reducingthe risk that pieces of the filter itself may dislodge and clog thechannel or otherwise interfere with device operation. Note that in thisembodiment, the filter has a gradient pore size (from large poresproximate the inlet to smaller pores proximal the narrowed portion ofthe flow channel); however, in alternative embodiments the pores may bethe same size or not gradient in size.

Further, in yet other embodiments the flow channel may not narrow. Inthese specific embodiments, the pores themselves can serve to providesuch a narrowing function for enhancing electroporation, and the flowchannel walls do not narrow or narrow minimally as the fluid flowsthrough the channel. These embodiments can allow control of the rate offlow of cells through the device to optimize introduction of nucleicacid into various cell types.

Moreover, though the scanning electromicrographs in FIG. 10E shown thefilter elements as rounded “pegs”, it should be understood that thefilter elements may be triangular-, square-, rectangular-, pentagonal-,hexagonal-, oval-, elliptical- or other faceted-shaped pegs.

FIG. 10F depicts (i) the electrodes 1008 before insertion into the FTEPdevice 1000 (here, a six-unit FTEP device) having inlet reservoirs 1052and outlet reservoirs 1054. In the preferred embodiment, the device 1000is used in an orientation inverted relative to that shown in FIG. 10F(i). FIG. 10F (ii) depicts an electrode 1008 contained within andprojecting from a sheath. FIG. 10F (iii) depicts the electrode 1008inserted into an electrode channel 1016 with the electrode channel 1016(and electrode 1008) adjacent to the flow channel 1006. In theembodiment shown in FIG. 10F (iii), the electrode is even with the wallsof the flow channel; that is, the electrode is not in the path of thecells/DNA/fluid flowing through flow channel 1006, however, neither isthe electrode recessed within the electrode channel 1016. Indeed, theelectrode 1008 may be recessed within the electrode channel 1016, may beextend to the end of electrode channel 1016 and thus be even with thewalls of flow channel 1006, or electrode 1008 may extend a minimaldistance into flow channel 1006 so long as the electrode does not impedemovement of the cells through the flow channel. The rounded or bevelededges of the aperture in the flow channel 1006 help prevent trapping airand reduce discontinuities in the electric field.

FIG. 10G presents two scanning electromicrographs of two differentconfigurations of the aperture where electrode channel 1016 meets flowchannel 1006. In FIG. 10G (i) (top), the edge of the junction ofelectrode channel 1016 and flow channel 1506 comprises a sharp edge. Incontrast, in FIG. 10G (ii) (bottom), the edges of the junction ofelectrode channel 1016 and flow channel 1006 comprises a rounded edge.Both configurations were tested (data not shown), and it was found thatthe rounded-edge configuration decreases the likelihood that air willbecome trapped between flow channel 1006 and the electrode (not seen inthis Figure) in electrode channel 1016. It can be seen that in thisembodiment the inlet apertures have a rounded edge, the advantages ofwhich include resistance to air trapping, promotion of laminar flow, andreduction of risk of cell damage. The rounded edges may have a radius ofcurvature of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or250 microns. Indeed, the electrodes of the FTEP devices should be “wet”;that is, immersed in the fluid/cells/DNA.

After transformation, the cells are allowed to recover under conditionsthat promote the transformation and/or expression of the introducednucleic acids in the cells. These temperatures and the duration ofmaintaining the temperatures can be determined by a preprogrammed set ofparameters (e.g., identified within the processing script or in anothermemory space accessible by the processing system), or manuallycontrolled by the user through interfacing with the processing system.

Once sufficient time has elapsed for the assembly reaction to takeplace, in some implementations, the nucleic acid assembly is transferredto a purification module. The processing system, for example, maymonitor timing of the assembly reaction based upon one or more of thetype of reaction, the type of materials, and user settings provided tothe automated instrument. The robotic handling system 1708 of FIGS.17A-17B, for example, may transfer the nucleic acid assembly to thepurification module through a sipper or pipettor interface. In anotherexample, the robotic handling system 1708 of FIGS. 17A-17B may transfera vial containing the nucleic acid assembly from a chamber of thenucleic acid assembly module to a chamber of the de-salt/purificationmodule.

In some implementations, the nucleic acid assembly is de-salted andeluted at the purification module. The purification module, for example,may remove unwanted components of the nucleic acid assembly mixture(e.g., salts, minerals, etc.). In some embodiments, the purificationmodule concentrates the assembled nucleic acids into a smaller volumethat the nucleic acid assembly volume. Examples of methods forexchanging liquid following nucleic acid assembly include magnetic beads(e.g., SPRI or Dynal (Dynabeads) by Invitrogen Corp. of Carlsbad,Calif.), silica beads, silica spin columns, glass beads, precipitation(e.g., using ethanol or isopropanol), alkaline lysis, osmoticpurification, extraction with butanol, membrane-based separationtechniques, filtration etc. For example, one or more micro-concentratorsfitted with anisotropic, hydrophilic-generated cellulose membranes ofvarying porosities may be used. In another example, thede-salt/purification module may process a liquid sample including anucleic acid and an ionic salt by contacting the mixture with an ionexchanger including an insoluble phosphate salt, removing the liquid,and eluting nucleic acid from the ion exchanger.

In an illustrative embodiment, the nucleic acid assembly may be combinedwith magnetic beads, such as SPRI beads, in a chamber of a purificationmodule. The nucleic acid assembly may be incubated at a set temperaturefor sufficient time for the assembled nucleic acids to bind to themagnetic beads. After incubation, a magnet may be engaged proximate tothe chamber so that the nucleic acid assembly can be washed and eluted.An illustrative example of this process is discussed in relation to thecombination nucleic acid assembly and purification module of FIG. 3.

Once the nucleic acid assembly has been eluted, the nucleic acidassembly, in some implementations, is transferred to the transformationmodule. The robotic handling system 1708 of FIGS. 17A-17B, for example,may transfer the assembled nucleic acids to the transformation modulethrough a sipper or pipettor interface to the FTEP as described above.For example, the de-salted assembled nucleic acids, during the transfer,may be combined with the electrocompetent cells from step 1408. In otherembodiments, the transformation module may accept each of theelectrocompetent cells and the nucleic acid assembly separately andenable the mixing (e.g., open one or more channels to combine thematerials in a shared chamber).

The cells are transformed in the FTEP module. A buffer or medium may betransferred to the transformation module and added to the cells so thatthe cells may be suspended in a buffer or medium that is favorable forcell survival during electroporation. Prior to transferring the bufferor medium, machine-readable indicia may be scanned upon the vial orother container or reservoir situated in the position designated for thebuffer or medium to confirm the contents of the vial, container, orreservoir. Further, the machine-readable indicia may indicate a type ofbuffer or medium provided to the instrument. The type of buffer ormedium, in some embodiments, may cause the instrument to select aparticular processing script (e.g., settings and activation of thetransformation module appropriate for the particular buffer or medium).For bacterial cell electroporation, low conductance mediums, such aswater or glycerol solutions, may be used to reduce the heat productionby transient high current. For yeast cells a sorbitol solution may beused. For mammalian cell electroporation, cells may be suspended in ahighly conductive medium or buffer, such as MEM, DMEM, IMDM, RPMI,Hanks', PBS, HBSS, HeBS and Ringer's solution. In a particular example,the robotic handling system may transfer a buffer solution to FTEPmodule from one of the cartridges. As described in relation to FIGS.4A-4I, 5A-5H, 6, 7A-7E, 8A-8U, and 9A-9C, the FTEP device may be adisposable FTEP device and/or the FTEP device may be provided with thecartridge (FTEP device 1124 of cartridge 1122 in FIG. 11E).

Once transformed, the cells are transferred to a second growth/recoverymodule. The robotic handling system 1708 of FIGS. 17A-17B, for example,may transfer the transformed cells to the second growth module through asipper or pipettor interface. In another example, the robotic handlingsystem 108 of 1A-1B or 1708 of FIGS. 17A-17B may transfer a vialcontaining the transformed cells from a chamber of the transformationmodule to a chamber of the second growth module.

The second growth module, in some embodiments, acts as a recoverymodule, allowing the cells to recover from the transformation process.In other embodiments, the cells may be provided to a separate recoverymodule prior to being transported to the second growth module. Duringrecovery, the second growth module allows the transformed cells touptake and, in certain aspects, integrate the introduced nucleic acidsinto the genome of the cell. The second growth module may be configuredto incubate the cells at any user-defined temperature optimal for cellgrowth, preferably 25°, 30°, or 37° C.

In some embodiments, the second growth module behaves as a selectionmodule, selecting the transformed cells based on an antibiotic or otherreagent. In the example of an antibiotic selection agent, the antibioticmay be added to the second growth module to enact selection. Suitableantibiotic resistance genes include, but are not limited to, genes suchas ampicillin-resistance gene, tetracycline-resistance gene,kanamycin-resistance gene, neomycin-resistance gene,canavanine-resistance gene, blasticidin-resistance gene,hygromycin-resistance gene, puromycin-resistance gene, orchloramphenicol-resistance gene. The robotic handling system 1708 ofFIGS. 17A-17B, for example, may transfer the antibiotic to the secondgrowth module through a sipper or pipettor interface. In someembodiments, removing dead cell background is aided using lyticenhancers such as detergents, osmotic stress by hyponic wash,temperature, enzymes, proteases, bacteriophage, reducing agents, orchaotropes. The processing system 1410 of FIG. 14, for example, mayalter environmental variables, such as temperature, to induce selection,while the robotic handling system 1708 of FIGS. 17A-17B may deliveradditional materials (e.g., detergents, enzymes, reducing agents, etc.)to aid in selection. In other embodiments, cell removal and/or mediaexchange by filtration is used to reduce dead cell background.

Some implementations may include the storage module 1714 of FIGS.17A-17B. The robotic handling system 1708 of FIGS. 17A-17B, for example,may transfer the cells to the storage module 114 through a sipper orpipettor interface. In another example, the robotic handling system 1708of FIGS. 17A-17B may transfer a vial containing the cells from a chamberof the second growth module to a vial or tube within the storage unit.

In some implementations, the method can be timed to introduce materialsand/or complete the transformation cycle or growth cycle in coordinationwith a user's schedule. For example, the automated instrument mayprovide the user the ability to schedule completion of one or more cellprocessing cycles such that the method is enacted with a goal ofcompletion at the user's preferred time. The time scheduling, forexample, may be set through a user interface, such as the user interface1416 of FIG. 14. For illustration only, a user may set completion of afirst cycle to 4:00 PM so that the user can supply additional cartridgesof materials to the automated instrument to enable overnight processingof another round of cell processing. Thus a user may time the programsso that two or more cycles may be programmed in a specific time period,e.g., a 24-hour period.

In some implementations, throughout the method, the automated instrumentmay alert the user to its current status. For example, the userinterface 1416 of FIG. 14 may present a graphical indication of thepresent stage of processing. In a particular example, a front face ofthe automated multi-module call processing instrument may be overlaidwith a user interface (e.g., touch screen) that presents an animatedgraphic depicting present status of the cell processing. The userinterface may further present any user and/or default settingsassociated with the current processing stage (e.g., temperature setting,time setting, etc.). In certain implementations, the status may becommunicated to a user via wireless communications controller.

Although illustrated as a particular series of operations, in otherembodiments, more or fewer steps may be included in the method. Forexample, in some embodiments, the contents of reservoirs, cartridges,and/or vials may be screened to confirm appropriate materials areavailable to proceed with processing. For example, in some embodiments,one or more imaging sensors (e.g., barcode scanners, cameras, etc.) mayconfirm contents at various locations within the housing of theautomated instrument. In one example, multiple imaging sensors may bedisposed within the housing of the automated instrument, each imagingsensor configured to detect one or more materials (e.g.,machine-readable indicia such as barcodes or QR codes, shapes/sizes ofmaterials, etc.). In another example, at least one imaging sensor may bemoved by the robotic handling system to multiple locations to detect oneor more materials. In further embodiments, one or more weight sensorsmay detect presence or absence of disposable or replaceable materials.In an illustrative example, the transfer tip supply holder may include aweight sensor to detect whether or not tips have been loaded into theregion. In another illustrative example, an optical sensor may detectthat a level of liquid waste has reached a threshold level, requiringdisposal prior to continuation of cell processing or addition of liquidif the minimum level has not been reached to proceed. Requests foradditional materials, removal of waste supplies, or other userinterventions (e.g., manual cleaning of one or more elements, etc.), insome implementations, are presented on a graphical user interface of theautomated instrument. The automated instrument, in some implementations,contacts the user with requests for new materials or other manualinterventions, for example through a software app, email, or textmessage.

Workflows for Cell Processing in an Automated Instrument

The automated instrument is designed to perform a variety of cellprocessing workflows using the same modules. For example, sourcematerials, in individual containers or in cartridge form, may differ andthe corresponding instructions (e.g., software script) may be selectedaccordingly, using the same basic instrumentation and robotic handlingsystem; that is, the instrument can be configured to perform a number ofdifferent workflows for processing cell samples and different types ofcell samples.

FIGS. 15A through 15C illustrate example workflows that may be performedusing an exemplary automated instrument including two cell growthmodules 1502, 1508, two filtration modules 1504 and 1510, and aflow-through electroporation module 1506. Although described as separategrowth modules 1502, 1508 and filtration modules 1504, 1510, each mayinstead be designed as a dual or integrated module. For example, a dualgrowth module, including growth modules 1502 and 1508, may include dualrotating growth vials sharing some circuitry, controls, and a powersource and disposed in a same housing. Similarly, a dual filtrationmodule may include filtration modules 1504 and 1510, including twoseparate filters and liquid supply tubes but sharing circuitry,controls, a power source, and a housing. The modules 1502, 1504, 1506,1508, and 1510, for example, may be part of the instrument.

Turning to FIG. 15A, a flow diagram illustrates a first workflow 1500involving processing steps resulting in introduction of nucleic acids toa cell stock 1512. This flow chart optionally uses a cartridge of sourcematerials. For example, a cartridge may include an oligo source 1514 aand a vector backbone 1516 a. The cell stock 1512, in some embodiments,is included with the oligo or reagents. The cell stock 1512 may besupplied within a kit including the cartridge. Alternatively, a user mayadd a container (e.g., vial or tube) of the cell stock 1512 to acartridge.

The workflow 1500, in some embodiments, is performed based upon a scriptexecuted by a processing system of the automated instrument, such as theprocessing system 1410 of FIG. 14. The script, in a first example, maybe accessed via a machine-readable marker or tag added to the firstcartridge. In some embodiments, each processing stage is performed usinga separate script. For example, each cartridge may include an indicationof a script or a script itself for processing the contents of thecartridge.

In some implementations, the first stage begins with introducing thecell stock 1512 into the first growth module 1502 for inoculation,growth, and monitoring (1518 a). In one example, a robotic handlingsystem adds a vial of the cell stock 1512 to medium contained in therotating growth vial of the first growth module 1502. In anotherexample, the robotic handling system pipettes cell stock 1512 from thefirst cartridge and adds the cell stock 1012 to the medium contained inthe rotating growth vial. The cells may have been maintained at atemperature of 4° C. at this point. In a particular example, 20 ml ofcell stock may be grown within a rotating growth vial of the firstgrowth module 1002 at a temperature of 30° C. to an OD of 0.50. The cellstock 1012 added to the first growth module 1502 may be monitored overtime until 0.50 OD is sensed via automated monitoring of the growthvial. Monitoring may be periodic or continuous. This may take, forexample, around 900 minutes (estimated), although the exact time dependsupon detection of the desired OD.

In some implementations, after growing the cells to the desired OD, aninducer is added to the first growth module 1502 for inducing the cells.In a particular example, 100 μl of inducer may be added, and the growthmodule 1502 may bring the temperature of the mixture up to 42° C. andhold for 15 minutes.

The cell stock 1512, after growth and induction, is transferred to thefirst filtration module 1504, in some implementations, for rendering thecells electrocompetent (1520 a) and to reduce the volume of the cellsfor transformation. In one example, a robotic handling system moves thevial of the cell stock 1512 from the rotating growth vial of the firstgrowth module 1502 to a vial holder of the first filtration module 1504.In another example, the robotic handling system pipettes cell stock 1512from the rotating growth vial of the first growth module 1502 anddelivers it to the first filtration module 1504. For example, thedisposable pipetting tip used to transfer the cell stock 1512 to thefirst growth module 1502 may be used to transfer the cell stock 1512from the first growth module 1502 to the first filtration module 1504.In some embodiments, prior to transferring the cell stock 1512 from thefirst growth module 1502 to the first filtration module 1504, the firstgrowth module 1502 is cooled to 4° C. so that the cell stock 1512 issimilarly reduced to this temperature. In a particular example, thetemperature of the first growth module 1502 may be reduced to about 4°C. over the span of about 8 minutes, and the growth module 1502 may holdthe temperature at 4° C. for about 15 minutes to ensure reduction intemperature of the cell stock 1512.

Prior to transferring the cell stock, in some implementations, a filterof the first filtration module 1504 is pre-washed using a wash solution.The wash solution, for example, may be supplied in a wash cartridge,such as the cartridge 1126 described in relation to FIG. 11C.

The first filtration module 1504, for example, may be part of a dualfiltration module such as the filtration module 1250 described inrelation to FIGS. 12B and 12C. In a particular example, the firstfiltration module 1504 may be maintained at 4° C. during the washing andeluting process while transferring cell materials between an elutionvial and the first filtration module 1504.

In some implementations, upon rendering the cells electrocompetent atthe filtration module 1504, the cell stock 1512 is transferred to atransformation module 1506 (e.g., flow-through electroporation module)for transformation. In one example, a robotic handling system moves thevial of the cell stock 1512 from the vial holder of the first filtrationmodule 1504 to a reservoir of the flow-through electroporation module1506. In another example, the robotic handling system pipettes cellstock 1512 from the first filtration module 1502 or a temporaryreservoir and delivers it to the first filtration module 1504. In aparticular example, 400 μl of the concentrated cell stock 1512 from thefirst filtration module 1504 is transferred to a mixing reservoir priorto transfer to the transformation module 1506. For example, the cellstock 1512 may be transferred to a reservoir in a cartridge for mixingwith the assembled nucleic acids, then transferred by the robotichandling system using a pipette tip. In a particular example, thetransformation module is maintained at 4° C.

While the cells are growing and/or rendered electrocompetent, in someimplementations, a first oligo source 1514 a and the vector backbone1516 a are assembled using a nucleic acid assembly process to createassembled nucleic acids, e.g., using a thermal cycler and ligationprocess or in an isothermal reaction within a nucleic acid assemblymodule (1522 a). The assembled nucleic acids may be created at somepoint during the first processing steps 1518 a, 1520 a of the firststage of the workflow 1500. Alternatively, assembled nucleic acids maybe created in advance of beginning the first processing step 1518.

In some embodiments, the nucleic acids are assembled using a nucleicacid assembly module of the automated instrument. For example, therobotic handling system may add the first oligo source 1514 a and avector backbone 1516 a from a vessel in the reagent cartridge in theautomated instrument to a nucleic acid assembly module (notillustrated), such as the nucleic acid assembly module 1710 g describedin relation to FIG. 17B. The nucleic acid assembly mix, for example, mayinclude in a particular example 50 μl Gibson Assembly® Master Mix, 25 μlvector backbone 1516 a, and 25 μl oligo 1514 a. The nucleic acidassembly module may be held at room temperature or at another desiredtemperature.

In other embodiments, the nucleic acids are assembled externally to theinstrument and added as a functioning source material. For example, avial or tube of assembled nucleic acids may be added to a reagentcartridge prior to activating the first step 1518 a of inoculation,growth and cell processing. In a particular example, 100 μl of assemblednucleic acids are provided.

In other embodiments, the nucleic acids are introduced to the cells incomponents, and the machinery of the transformed cells will perform theassembly within the cell, e.g., using gap repair mechanisms in yeastcells.

In some implementations, the assembled nucleic acids are purified (1524a). The assembled nucleic acids, for example, may be transferred by therobotic handling system from the nucleic acid assembly module to apurification module (not shown). In other embodiments, the nucleic acidassembly module may include purification features (e.g., a combinationnucleic acid assembly and purification module). In further embodiments,the assembled or separate nucleic acids are purified externally to theinstrument and added as a functional source material. For example, avial or tube of purified assembled nucleic acids may be added to areagent cartridge with the cell stock 1012 prior to activating the firststep 1518 a of cell processing.

In a particular example, 100 μl of assembled nucleic acids in nucleicacid assembly mix are assembled and subsequently purified. In someembodiments, magnetic beads are added to the nucleic acid assemblymodule, for example 180 μl of magnetic beads in a liquid suspension maybe added to the nucleic acid assembly module by the robotic handlingsystem. A magnet functionally coupled to the nucleic acid assemblymodule may be activated and the sample washed in 200 μl ethanol (e.g.,the robotic handling system may transfer ethanol to the nucleic acidassembly module). Liquid waste from this operation, in some embodiments,is transferred to a waste receptacle of the cartridge (e.g., by therobotic handling system using a same pipette tip as used in transferringthe ethanol). At this point, the de-salted assembled nucleic acids maybe transferred to a holding container, such as a reservoir of thecartridge. The desalted assembled nucleic acids may be held, for exampleat a temperature of 4° C. until cells are ready for transformation. In aparticular example, 100 μl of the assembled nucleic acids may be addedto the 400 μl of the concentrated cell stock 1512 in the mixingreservoir prior to transfer to the transformation module 1506. In someembodiments, the purification process may take about 16 minutes.

In some implementations, the assembled nucleic acids and cell stock 1512are added to the flow-through electroporation module 1506 and the cellstock 1512 is transformed (1526 a). The robotic handling system, forexample, may transfer the mixture of the cell stock 1512 and assemblednucleic acids to the flow-through electroporation module 1506 from amixing reservoir, e.g., using a pipette tip or through transferring avial or tube. In some embodiments, a built-in flow-throughelectroporation module such as the flow-through electroporation modulesdepicted in FIGS. 4A-4I, 5A-5H, 6, 8A-8U, and 9A-9C is used to transformthe cell stock 1512. In other embodiments, a cartridge-basedelectroporation module such as shown in FIGS. 10A-10C and 10E is used totransform the cell stock 1512. The electroporation module 1506, forexample, may be held at a temperature of 4° C. The electroporationprocess, in an illustrative example, may take about four minutes.

The transformed cell stock 1512 in some implementations is transferredto the second growth module 1508 for recovery (1528 a). In a particularexample, transformed cells undergo a recovery process in the secondgrowth module 1508 at a temperature of 30° C. The transformed cells, forexample, may be maintained in the second growth module 1508 for apredetermined period of time, e.g., about an hour for recovery.

In some implementations, a selective medium is transferred to the secondgrowth vial (not illustrated), and the cells are left to incubate for afurther period of time in a selection process. In an illustrativeexample, an antibiotic may be transferred to the second growth vial, andthe cells may incubate for an additional two hours at a temperature of30° C. to select for cells that have received the nucleic acids.

In some implementations, in preparation for further processing, thetransformed cells are transferred to the second filtration module 1510for media exchange and filtering (1530 a). Prior to transferring thetransformed cell stock, in some implementations, a filter of the secondfiltration module 1504 is pre-washed using a wash solution. The washsolution, for example, may be supplied in a wash cartridge, such as thecartridge 1126 described in relation to FIG. 11C. The second filtrationmodule 1510, for example, may be fluidly connected to the wash solutionof the wash cartridge, as described in relation to FIG. 12A.

The second filtration module 1510, for example, may be part of a dualfiltration module such as the filtration module 1250 described inrelation to FIGS. 12B and 12C. In a particular example, the secondfiltration module 1510 may be maintained at a predetermined temperature(e.g., 4° C.) during the washing and eluting process while transferringcell materials between an elution vial and the second filtration module1510. The output of this filtration process, in a particular example, isdeposited in a vial or tube to await further processing, e.g., transferto a transformation module. The vial or tube may be maintained in astorage module at a temperature of 4° C.

In other implementations, turning to FIG. 15B, a workflow 1540 involvesthe same modules 1502, 1504, 1506, 1508, and 1510 as well as the sameprocessing steps 1518, 1520, 1522, 1524, 1526, 1528, and 1530 for thefirst stage of process. However, unlike the workflow 1500 of FIG. 15A,the workflow 1540 of FIG. 15B includes the additional steps of inductionand/or concentration of cells of interest (i.e. transformed cells) andstorage of the cells once selected and/or concentrated. For example, thecells may undergo selection in a cell selection/concentration module1532, followed by storage of the selected/concentrated cells in astorage module 1534. In certain implementations, the cellselection/concentration module and storage module are combined into asingle, integrated module.

As with the workflow 1500, in some embodiments, the workflow 1540 isperformed based upon a script executed by a processing system of theautomated instrument, such as the processing system 1410 of FIG. 14. Thescript, in a first example, may be accessed via a machine-readablemarker or tag added to the first cartridge. In some embodiments, eachprocessing stage is performed using a separate script. For example, eachcartridge may include an indication of a script or a script itself forprocessing the contents of the cartridge. The automated instrument inthis implementation includes a n oligo source, the cells are inoculated,grown, and monitored in the first growth module 1502 (1518 d). In aparticular example, an aliquot of the cell stock 1542 may be transferredto a rotating growth vial containing, e.g., 20 mL of growth medium at atemperature of 30° C. to an OD of 0.50. The cell stock 1542 added to thefirst growth module 1502 may be monitored over time until 0.50 OD issensed via the automated monitoring. Monitoring may be periodic orcontinuous. This may take, for example, around 900 minutes (estimated),although the exact time depends upon detection of the desired OD.

In some implementations, after growing to the desired OD, an inducer isadded to the first growth module 1502 for inducing the cells. In aparticular example, 100 μl of inducer may be added, and the growthmodule 1502 may bring the temperature of the mixture up to 42° C. andhold for 15 minutes.

The cell stock 1542, after growth and induction, is transferred to thefirst filtration module 1504, in some implementations, for rendering thecells electrocompetent (1520 d). In one example, a robotic handlingsystem moves the vial of the cell stock 1542 from the rotating growthvial of the first growth module 1502 to a vial holder of the firstfiltration module 1504. In another example, the robotic handling systempipettes cell stock 1542 from the rotating growth vial of the firstgrowth module 1502 and delivers it to the first filtration module 1504.For example, the disposable pipetting tip used to transfer the cellstock 1542 to the first growth module 1502 may be used to transfer thecell stock 1542 from the first growth module 1502 to the firstfiltration module 1504. In some embodiments, prior to transferring thecell stock 1542 from the first growth module 1502 to the firstfiltration module 1504, the first growth module 1502 is cooled to 4° C.so that the cell stock 1542 is similarly reduced to this temperature. Ina particular example, the temperature of the first growth module 1502may be reduced to about 4° C. over the span of about 8 minutes, and thegrowth module 1502 may hold the temperature at 4° C. for about 15minutes to ensure reduction in temperature of the cell stock 1512.

Prior to transferring the cell stock 1542 to the filtration module, insome implementations a filter of the first filtration module 1504 ispre-washed using a wash solution. The wash solution, for example, may besupplied in a wash cartridge, such as the cartridge 1126 described inrelation to FIG. 11C. The first filtration module 1504, for example, maybe fluidly connected to the wash solution of the wash cartridge.

The first filtration module 1504, for example, may be part of a dualfiltration module such as the filtration module 1250 described inrelation to FIGS. 12B and 12C. In a particular example, the firstfiltration module 1504 may be maintained at a predetermined temperature(e.g., 4° C.) during the washing and eluting process while transferringcell materials between an elution vial and the first filtration module1504.

In some implementations, upon rendering the cells electrocompetent atthe filtration module 1504 (1520 d), the cell stock 1542 is transferredto a transformation module 1506 (e.g., FTEP module) for transformation.In one example, a robotic handling system moves the vial of the cellstock 1542 from the vial holder of the first filtration module 1504 to areservoir of the flow-through electroporation module 1506. In anotherexample, the robotic handling system pipettes cell stock 1542 from thefirst filtration module 1502 or a temporary reservoir and delivers it tothe first filtration module 1504. In a particular example, 400 μl of theconcentrated cell stock 1542 from the first filtration module 1504 istransferred to a mixing reservoir prior to transfer to thetransformation module 1506. For example, the cell stock 1542 may betransferred to a reservoir in a cartridge, then mixed and transferred bythe robotic handling system using a pipette tip. In a particularexample, the transformation module 1506 is maintained at a predeterminedtemperature, e.g., 4° C. The cell stock 1542 may be transformed, in anillustrative example, in about four minutes.

The transformed cell stock 1542, in some implementations, is transferredto the second growth module 1508 for recovery (1528 d). In a particularexample, 20 ml of transformed cells undergo a recovery process in thesecond growth module 1508 at a temperature of 30° C. The transformedcells, for example, may be maintained in the second growth module 1508for about an hour for recovery.

After recovery, the cells may be ready for further processing (e.g.,induction of protein expression or cell sorting) or for storage to beused in further research outside the automated cell processinginstrument. For example, a portion of the cells may be transferred to astorage module as cell library output, while another portion of thecells may be prepared for a protein expression and isolation.

In some implementations, in preparation for further processing or forcell concentration, the transformed cells are transferred to the secondfiltration module 1510 for media exchange and filtering (1530 d)containing glycerol for rendering the cells electrocompetent. Prior totransferring the transformed cell stock, in some implementations, afilter of the second filtration module 1504 is pre-washed using a washsolution. The wash solution, for example, may be supplied in a washcartridge. The second filtration module 1510, for example, may befluidly connected to the wash solution of the wash cartridge, asdescribed in relation to FIG. 12A.

The second filtration module 1510, for example, may be part of a dualfiltration module such as the filtration module 1250 described inrelation to FIGS. 12B and 12C. In a particular example, the secondfiltration module 1510 may be maintained at 4° C. during the washing andeluting process while transferring cell materials between an elutionvial and the second filtration module 1510. The output of thisfiltration process, in a particular example, are electrocompetent cellsdeposited in a vial or tube to await further processing. The vial ortube may be maintained in a storage module at a temperature of 4° C.

Turning to FIG. 15C, a flow diagram illustrates another workflow 1560involving an induction of protein expression from the introduced oligosand isolation of the produced proteins. The workflow 1560, in someembodiments, is performed based upon a script executed by a processingsystem of the automated instrument, such as the processing system 1410of FIG. 14. The script, in a first example, may be accessed via amachine-readable marker or tag added to the first cartridge. In someembodiments, each processing stage is performed using a separate script.For example, each cartridge may include an indication of a script or ascript itself for processing the contents of the cartridge.

In some implementations, the first stage begins with introducing thecell stock 1562 into the first growth module 1502 for inoculation,growth, and monitoring (1518 e). In one example, a robotic handlingsystem adds a vial of the cell stock 1562 to a rotating growth vial ofthe first growth module 1502. In another example, the robotic handlingsystem pipettes cell stock 1562 from the first cartridge and adds thecell stock 1562 to the rotating growth vial. The cells may have beenmaintained at a temperature of 4° C. at this point. In a particularexample, 20 ml of cell stock may be grown within a rotating growth vialof the first growth module 1502 at a temperature of 30° C. to an OD of0.75. The cell stock 1512 added to the first growth module 1502 may beautomatically monitored over time within the growth module 1502 until0.75 OD is sensed via the automated monitoring. Monitoring may beperiodic or continuous.

In some implementations, an inducible expression system may be used.Thus, after growing to the desired OD, an inducer is added to the firstgrowth module 1502 for inducing protein production the cells. Theinducer could be a small molecule or a media exchange to a medium with adifferent sugar like galactose.

The cell stock 1562, after growth and induction, is transferred to thefirst filtration module 1504, in some implementations, for exchangingmedia (1564 a). In one example, a robotic handling system moves the vialof the cell stock 1562 from the rotating growth vial of the first growthmodule 1502 to a vial holder of the first filtration module 1504. Inanother example, the robotic handling system pipettes cell stock 1562from the rotating growth vial of the first growth module 1502 anddelivers it to the first filtration module 1504. For example, thedisposable pipetting tip used to transfer the cell stock 1562 a to thefirst growth module 1502 may be used to transfer the cell stock 1562from the first growth module 1502 to the first filtration module 1504.In some embodiments, prior to transferring the cell stock 1562 from thefirst growth module 1502 to the first filtration module 1504, the firstgrowth module 1502 is cooled to 4° C. so that the cell stock 1562 issimilarly reduced to this temperature. In a particular example, thetemperature of the first growth module 1502 may be reduced to about 4°C. over the span of about 8 minutes, and the growth module 1502 may holdthe temperature at 4° C. for about 15 minutes to ensure reduction intemperature of the cell stock 1562. During media exchange, in anillustrative example, 0.4 ml of 1M sorbitol may be added to the cellstock 1562.

Prior to transferring the cell stock 1562, in some implementations, afilter of the first filtration module 1004 is pre-washed using a washsolution. The wash solution, for example, may be supplied in a washcartridge. The first filtration module 1504, for example, may be fluidlyconnected to the wash solution of the wash cartridge, as described inrelation to FIG. 12A.

The first filtration module 1504, for example, may be part of a dualfiltration module such as the filtration module 1250 described inrelation to FIGS. 12B and 12C. In a particular example, the firstfiltration module 1504 may be maintained at 4° C. during the washing andeluting process while transferring cell materials between an elutionvial and the first filtration module 1504.

After the media exchange operation, in some implementations, the cellstock 1562 is transferred back to the first growth module 1502 forconditioning (1566 a). In one example, a robotic handling system movesthe vial of the cell stock 1562 from the first filtration module 1504 tothe first growth module 1502. In another example, the robotic handlingsystem pipettes cell stock 1562 from the first filtration module 1504and delivers it to the rotating growth vial of the first growth module1502. During conditioning, for example, 5 ml DTT/LIAc and 80 mM ofSorbitol may be added to the cell stock 1562. For example, the robotichandling system may transfer the DTT/LIAc and Sorbitol, individually orconcurrently, to the first growth module 1502. The cell stock 1562 maybe mixed with the DTT/LIAc and Sorbitol, for example, via the rotationof the rotating growth vial of the first growth module 1502. Duringconditioning, the cell stock 1562 may be maintained at a temperature of4° C.

In some implementations, after conditioning, the cell stock 1562 istransferred to the first filtration module 1504 for washing andpreparing the cells (1568). For example, the cells may be renderedelectrocompetent at this step. In one example, a robotic handling systemmoves the vial of the cell stock 1562 from the rotating growth vial ofthe first growth module 1502 to a vial holder of the first filtrationmodule 1504. In another example, the robotic handling system pipettescell stock 1562 from the rotating growth vial of the first growth module1502 and delivers it to the first filtration module.

Prior to transferring the cell stock, in some implementations, a filterof the first filtration module 1504 is pre-washed using a wash solution.The wash solution, for example, may be supplied in a wash cartridge. Thefirst filtration module 1504, for example, may be fluidly connected tothe wash solution of the wash cartridge, as described in relation toFIG. 12A. In other embodiments, the same filter is used for renderingelectrocompetent as the filter used for media exchange at step 1564 a.In some embodiments, 1M sorbitol is used to render the yeast cellselectrocompetent.

In some implementations, upon rendering electrocompetent at thefiltration module 1504, the cell stock 1562 is transferred to atransformation module 1506 (e.g., flow-through electroporation module)for transformation. In one example, a robotic handling system moves thevial of the cell stock 1562 from the vial holder of the first filtrationmodule 1504 to a reservoir of the flow-through electroporation module1506. In another example, the robotic handling system pipettes cellstock 1562 from the filtration module 1504 or a temporary reservoir anddelivers it to the first filtration module 1504. In a particularexample, 400 μl of the concentrated cell stock 1562 from the firstfiltration module 1504 is transferred to a mixing reservoir prior totransfer to the transformation module 1506. For example, the cell stock1562 may be transferred to a reservoir in a cartridge for mixing withthe nucleic acid components (vector backbone and oligonucleotide), thenmixed and transferred by the robotic handling system using a pipettetip. Because the vector backbone and oligonucleotide are assembled inthe cells (in vivo), a nucleic acid assembly module is not a necessarycomponent. In a particular example, the transformation module ismaintained at 4° C.

In some implementations, the nucleic acids to be assembled and the cellstock 1562 is added to the FTEP module 1506 and the cell stock 1562 istransformed (1526 e). The robotic handling system, for example, maytransfer the mixture of the cell stock 1562 e and nucleic acid assemblyto the flow-through electroporation module 1506 from a mixing reservoir,e.g., using a pipette tip or through transferring a vial or tube. Insome embodiments, a built-in FTEP module such as the flow-throughelectroporation modules FIGS. 4A-4I, 5A-5G, 6, 8A-8U, 9A-9C, and 10A-10C(that is, single unit FTEPs) is used to transform the cell stock 1562 e.In other embodiments, a cartridge-based electroporation module is usedto transform the cell stock 1562 e. The FTEP module 1506, for example,may be held at a temperature of 4° C.

The transformed cell stock 1562 e, in some implementations, istransferred to the second growth module 1508 for recovery (1528 a). In aparticular example, 20 ml of transformed cells undergo a recoveryprocess in the second growth module 1508.

In some implementations, a selective medium, e.g. an auxotrophic growthmedium or a medium containing a drug, is transferred to the secondgrowth vial (not illustrated), and the cells are left to incubate for afurther period of time in a selection process. In an illustrativeexample, an antibiotic may be transferred to the second growth vial, andthe cells may incubate for an additional two hours at a temperature of30° C.

After recovery, the cells may be ready for further processing or forstorage in a cell library. For example, a portion of the cells may betransferred to a storage module as cell library output (1576 a), whileanother portion of the cells may be prepared for processing, e.g.,induction of protein expression and isolation of produced proteins (1578a). The cells may be stored, for example, at a temperature of 4° C.

In some implementations, in preparation for processing, the transformedcells are transferred to the second filtration module 1510 for mediaexchange (1578 a). Prior to transferring the transformed cell stock 1562a, in some implementations, a filter of the second filtration module1504 is pre-washed using a wash solution. The wash solution, forexample, may be supplied in a wash cartridge. The second filtrationmodule 1510, for example, may be fluidly connected to the wash solutionof the wash cartridge, as described in relation to FIG. 12A.

The second filtration module 1510, for example, may be part of a dualfiltration module such as the filtration module 1250 described inrelation to FIGS. 12B and 12C. In a particular example, the secondfiltration module 1510 may be maintained at 4° C. during the washing andeluting process while transferring cell materials between an elutionvial and the second filtration module 1510.

In some implementations during the filtration process, an enzymaticpreparation is added to lyse the cell walls of the cell stock 1562 a.For example, a yeast lytic enzyme such as Zylomase® may be added to lysethe cell walls. The yeast lytic enzyme, in a particular example, may beincubated in the cell stock 1526 a for between 5-60 minutes at atemperature of 30° C. The output of this filtration process, in aparticular example, is deposited in a vial or tube to await furtherprocessing. The vial or tube may be maintained in a storage module at atemperature of 4° C.

The first stage of processing may take place during a single day. Atthis point of the workflow 1560, in some implementations, new materialsare manually added to the automated instrument. For example, new cellstock 1562 b and a new reagent cartridge may be added. Further, a newwash cartridge, replacement filters, and/or replacement pipette tips maybe added to the automated instrument at this point. Further, in someembodiments, the filter module may undergo a cleaning process and/or thesolid and liquid waste units may be emptied in preparation for the nextround of processing.

FIGS. 17A and 17B illustrate embodiments of automated multi-module cellprocessing instruments for performing cell transformation, selection,gene analysis, or protein expression. The automated multi-module cellprocessing instruments, for example, may be desktop instruments designedfor use within a laboratory environment. The automated multi-module cellprocessing instruments may incorporate both reusable and disposableelements for performing various staged operations in conductingautomated genome cleavage and/or protein production in cells.

FIG. 17A is a block diagram of a first example instrument 1700 forperforming automated cell processing. In some implementations, theinstrument 1700 includes a deck, a reagent supply receptacle 1704 forintroducing DNA sample components to the instrument 1700, a cell supplyreceptacle 1706 for introducing cells to the instrument 1700, and arobot handling system 1708 for moving materials between modules (forexample, modules 1710 a, 1710 b, 1710 c, 1710 d) receptacles (forexample, receptacles 1704, 1706, 1712 a-c, 1722, 1724, and 1726), andstorage units (e.g., units 1718, 1728, and 1714) of the instrument 1700to perform the automated cell processing. Upon completion oftransformation of the cell supply 1706, in some embodiments, cell output1712 may be transferred by the robot handling system 1708 to a storageunit 1714 for temporary storage and later retrieval.

The robotic handling system 1708, for example, may include an airdisplacement pump to transfer liquids from the various material sourcesto the various modules 1710 a-d and storage unit 1714. In otherembodiments, the robotic handling system 1708 may include a pick andplace head to transfer containers of source materials (e.g., tubes) froma supply cartridge (not illustrated, discussed in relation to FIG. 1A)to the various modules 1710 a-d. In some embodiments, one or morecameras or other optical sensors (not shown), confirm proper gantrymovement and position.

In some embodiments, the robotic handling system 1708 uses disposabletransfer tips provided in a transfer tip supply 1716 to transfer sourcematerials, reagents 1704 (e.g., for nucleic acid assembly), and cells1706 within the instrument 1700. Used transfer tips, for example, may bediscarded in a solid waste unit 1718. In some implementations, the solidwaste unit 1718 contains a kicker to remove tubes from the pick andplace head of robotic handling system 1708.

In some implementations, the instrument 1700 is controlled by aprocessing system 1720 such as the processing system 1410 of FIG. 14.The processing system 1720 may be configured to operate the instrument1700 based on user input. The processing system 1720 may control thetiming, duration, temperature and other operations of the variousmodules 1710 of the instrument 1700. The processing system 1720 may beconnected to a power source (not shown) for the operation of theinstrument 1700.

Instrument 1700 includes an FTEP device 1710 c to introduce nucleicacid(s) into the cells 1706. For example, the robotic handling system1708 may transfer the reagent 1704 and cells 1706 to the FTEP device1710 c. The FTEP device 1710 conducts cell transformation ortransfection via electroporation. The processing system 1720 may controltemperature and operation of the FTEP device 1710 c. In someimplementations, the processing system 1720 effects operation of theFTEP device 1710 c according to one or more variable controls set by auser.

Following transformation, in some implementations, the cells may betransferred to a recovery module 1710 d. In some embodiments, therecovery module 1710 d is a combination recovery and induction ofprotein production module. In the recovery module 1710 d, the cells areallowed to recover, express the nucleic acids and, in an induciblesystem, transcription of the introduced nucleic acids is induced in thecells, e.g., by means of temporally-controlled induction such as, insome examples, chemical, light, viral, or temperature induction or theintroduction of an inducer molecule 1724 for expression of the protein.

Following protein production, in some implementations the cells aretransferred to the storage unit 1714, where the cells can be stored ascell output 1712 a-d until the cells are removed for further study orretrieval of a transformed cell population, e.g., a transformed celllibrary.

A portion of a cell output 1712 a, in some embodiments, is transferredto an automated cell growth module 1710 a. For example, all of the celloutput 1712 a may be transferred, or only an aliquot may be transferredsuch that the instrument retains incrementally modified samples. Thecell growth module 1710 a, in some implementations, measures the OD ofthe cells during growth to ensure they are at a desired concentrationprior to induction of transcription and, in some aspects, translation.Other measures of cell density and physiological state that can be usedinclude but are not limited to, pH, dissolved oxygen, released enzymes,acoustic properties, and electrical properties.

To reduce the background of cells that have not been transformed, insome embodiments the growth module 1710 a performs a selection processto enrich for the transformed cells using a selective growth medium1726. For example, the introduced nucleic acid can include a gene thatconfers antibiotic resistance or some other selectable marker. In someimplementations, multiple selective genes or markers 1726 may beintroduced into the cells during processing. Suitable antibioticresistance genes include, but are not limited to, genes such asampicillin-resistance gene, tetracycline-resistance gene,kanamycin-resistance gene, neomycin-resistance gene,canavanine-resistance gene, blasticidin-resistance gene,hygromycin-resistance gene, puromycin-resistance gene, andchloramphenicol-resistance gene.

From the growth module 1710 a, the cells may be transferred to afiltration module 1710 b. The filtration module 1710 b or,alternatively, a cell wash and concentration module, may enable mediaexchange. In some embodiments, removing dead cell background is aidedusing lytic enhancers such as detergents, osmotic stress, temperature,enzymes, proteases, bacteriophage, reducing agents, or chaotropes. Inother embodiments, cell removal and/or media exchange is used to reducedead cell background. Waste product from the filtration module 1710 b,in some embodiments, is collected in a liquid waste unit 1728.

After filtration, the cells may be presented to the FTEP device(transformation module) 1710 c, and then to the recovery module 1710 dand finally to the storage unit 1714 as detailed above.

Turning to FIG. 17B, similar to FIG. 17A, a second exemplary instrument1740 for performing automated genome cleavage and/or protein productionin cells, the reagent supply receptacle 1704 for introducing one or morenucleic acid components to the instrument 1740, the cell supplyreceptacle 1706 for introducing cells to the instrument 1740, and therobot handling system 1708 for moving materials between modules (forexample, modules 1710 a, 1710 b, 1710 c, 1710 f, 1710 g, 1710 m, and1710 h), receptacles (for example, receptacles 1704 1706, 1712 a-c,1724, 1742, 1744, and 1746), and storage units (e.g., units 1714, 1718,and 1728) of the instrument 1740 to perform the automated cellprocessing. Upon completion of processing of the cell supply 1706, insome embodiments, cell output 1712 a-c may be transferred by the robothandling system 1708 to the storage unit 1714 for temporary storage andlater retrieval.

In some embodiments, the robotic handling system 1708 uses disposabletransfer tips provided in the transfer tip supply 1716 to transfersource materials, a vector backbone 1742, an expression cassette oroligos 1744, reagents 1704 (e.g., for nucleic acid assembly, nucleicacid purification, to render cells electrocompetent, etc.), and cells1706 within the instrument 1740, as described in relation to FIG. 17A.

As described in relation to FIG. 17A, in some implementations, theinstrument 1740 is controlled by the processing system 1720 such as theprocessing system 1410 of FIG. 14.

The instrument 1740, in some embodiments, includes a nucleic acidassembly module 1710 g, and in certain exemplary automated multi-modulecell processing instruments, the nucleic acid assembly module 1710 g mayperform in some embodiments nucleic acid assembly.

In some embodiments, after assembly of the nucleic acids, the nucleicacids (e.g., in the example of a nucleic acid assembly, the nucleic acidassembly mix (nucleic acids+nucleic acid assembly reagents)) aretransferred to a purification module 1710 h. Here, unwanted componentsof the nucleic acid assembly mixture are removed (e.g., salts) and, incertain embodiments, the assembled nucleic acids are concentrated. Forexample, in an illustrative embodiment, in the purification module 1710h, the nucleic acid assembly mix may be combined with a no-salt bufferand magnetic beads, such as Solid Phase Reversible Immobilization (SPRI)magnetic beads or AMPure™ beads. The nucleic acid assembly mix may beincubated for sufficient time (e.g., 30 seconds to 10 minutes) for theassembled nucleic acids to bind to the magnetic beads. In someembodiments, the purification module includes a magnet configured toengage the magnetic beads. The magnet may be engaged so that thesupernatant may be removed from the bound assembled nucleic acids and sothat the bound assembled nucleic acids can be washed with, e.g., 80%ethanol. Again, the magnet may be engaged and the 80% ethanol washsolution removed. The magnetic bead/assembled nucleic acids may beallowed to dry, then the assembled nucleic acids may be eluted and themagnet may again be engaged, this time to sequester the beads and toremove the supernatant that contains the eluted assembled nucleic acids.The assembled nucleic acids may then be transferred to thetransformation module (e.g., electroporator in a preferred embodiment).The transformation module may already contain the electrocompetent cellsupon transfer.

Instrument 1740 includes an FTEP device 1710 c for introduction of thenucleic acid(s) into the cells 1706, as described in relation to FIG.17A. However, in this circumstance, the assembled nucleic acids 1704,output from the purification module 1710 h, are transferred to the FTEPdevice 1710 c to combine with the cells 1706.

Following transformation in the FTEP device 1710 c, in someimplementations, the cells may be transferred to a recovery module 1710m. In the recovery module 1710 e, the cells are allowed to recover,express the exogenous nucleic acids electroporated into the cells and,in an inducible system, transcription and translation of a protein isinduced, e.g., by means of temporally-controlled induction such as, insome examples, chemical, light, viral, or temperature induction or theintroduction of the inducer molecule for expression of the protein.

Following recovery, in some implementations the cells are transferred toan expression module 1710 f. The expression module 1710 f providesappropriate conditions to induce production of a protein, e.g., throughexpression of the introduced nucleic acids and the induction of aninducible protein. The protein may be, in some examples, chemicallyinduced, biologically induced (e.g., via inducible promoter) virallyinduced, light induced, temperature induced, and/or heat induced withinthe expression module 1710 f.

Following transformation (and, e.g., protein production), in someimplementations, the cells are transferred to the storage unit 1714 asdescribed in relation to FIG. 17A.

A portion of a cell output 1712 a, in some embodiments, is transferredto the automated cell growth module 1710 a, as discussed in relation toFIG. 17A.

To reduce background of cells that have not been transformed, in someembodiments, the growth module 1710 a performs a selection process toenrich for the transformed cells using a selective growth medium 1726,as discussed in relation to FIG. 17A.

From the growth module 1710 a, the cells may be transferred to thefiltration module 1710 b, as discussed in relation to FIG. 17A. Asillustrated, eluant from an eluting supply 1746 (e.g. buffer, glycerol)may be transferred into the filtration module 1710 b for media exchange.

After filtration, the cells may be transferred to the FTEP device 1710 cfor transformation, and then to the recovery module 1710 m, and theprotein expression module 1710 f and finally to the storage unit 1714 asdetailed above.

In some embodiments, the automated multi-module cell processinginstruments of FIGS. 17A and/or 17B contain one or more replaceablesupply cartridges and a robotic handling system. Each cartridge maycontain one or more of a nucleic acid assembly mix, oligonucleotides,vector, growth media, selection agent (e.g., antibiotics), inducingagent, nucleic acid purification reagents such as Solid Phase ReversibleImmobilization (SPRI) beads, ethanol, and 10% glycerol.

Although the exemplary instruments 1700, 1740 are illustrated asincluding a particular arrangement of modules 1710, these arrangementsare for illustrative purposes only. For example, in other embodiments,more or fewer modules 1710 may be included within each of theinstruments 1700, 1740. Also, different modules may be included in theinstrument, such as, e.g., a module that facilitates cell fusion forproviding, e.g., hybridomas, a module that amplifies nucleic acidsbefore assembly, and/or a module that facilitates protein production.Further, certain modules 1710 may be replicated within certainembodiments, such as the duplicate cell growth modules. Each of theinstruments 1700 and 1740, in another example, may be designed to accepta media cartridge such as the cartridges of FIGS. 11A and 11C. Furthermodifications are possible.

Protein Expression Module

Alternatively, or in addition, the instrument may include a proteinexpression module where cells are allowed to express the nucleic acidsintroduced by transformation of the cells in the system. Traditionalstrategies for recombinant protein expression involve culturing thetransformed cells so that they transcribe and translate the desiredprotein. Typically, the cells are then lysed to extract the expressedprotein for subsequent purification, and such lysis can be performed onthe instrument or following collection of the cells from the system.Both prokaryotic and eukaryotic in vivo protein expression systems maybe used in the instruments of the disclosure.

The selection of the system depends on the type of protein, therequirements for functional activity and the desired yield. Theexpression systems that can be used with the instruments of thedisclosure include any expression system known to a person skilled inthe art amenable to automated transformation or transfection, includingas eukaryotic expression systems such as yeasts (S. cerevisiae or P.pastoris), bacterial expression systems, insect cells (sf9) or mammalianexpression systems such as CHO, 293 or HEK cells. In one embodiment, itis preferred to transform the vector in an E. coli expression system,wherein E. coli BL21(DE3) is particularly preferred. Each system hasadvantages and challenges, and the particular system that can be used inthe inventions of the disclosure can be selected for the particularapplication, as will be apparent to one of ordinary skill in the artupon reading the present disclosure.

Cell Sorting Module

Alternatively, or in addition, the instrument may include a sortingmodule where cells expressing different cell surface markers are sortedfrom those cells that do not express such cell surface markers. Forexample, fluorescence-activated cell sorting (“FACS”) e.g., differentfluorophores or other optically-distinguishable markers that bind to thecell surface markers of interest are used to sort cells. Fluorophores ofuse in this aspect include TagBFP, TagCFP, TagGFP2, TagYFP, TagRFP,FusionRed, mKate2, TurboGFP, TurboYFP, TurboRFP, TurboFP602, TurboFP635,TurboFP650, AmCyan1, AcvGFP1, ZsGreen1, ZsYellow1, mBanana, mOrange,mOrange2, DsRed-Express2, EsRed-Express, tdTomato, DsRed-Monomer,DsRed2, AsRed2, mStrawberry, mCherry, HcRed1, mRaspberry, E2-Crimson,mPlum, Dendra 2, Timer, and PAmCherry, HALO-tags, or infrared-shiftedfluorescent proteins. Alternatively, chemiluminescent markers may beemployed.

Control System for an Automated Instrument

Turning to FIG. 16, a screen shot illustrates an example graphical userinterface (GUI) 1600 for interfacing with an automated instrument. Theinterface, for example, may be presented on the display 236 of FIGS. 2Cand 2D. In one example, the GUI 1600 may be presented by the processingsystem 1410 of FIG. 14 on the touch screen 1416.

In some implementations, the GUI 1600 is divided into a number ofinformation and data entry panes, such as a protocol pane 1602, atemperature pane 1606, an electroporation pane 1608, and a cell growthpane 1610. Further panes are possible. For example, in some embodimentsthe GUI 1600 includes a pane for each module, such as, in some examples,one or more of each of a nucleic acid assembly module, a purificationmodule, a cell growth module, a filtration module, a transformationmodule, and a recovery module. The lower panes of the GUI 1600, in someembodiments, represent modules applicable to the present work flow(e.g., as selected in the protocol pane 1602 or as designated within ascript loaded through a script interface (not illustrated)). In someembodiments, a scroll or paging feature may allow the user to accessadditional panes not illustrated within the screen shot of FIG. 16.

The GUI 1600, in some embodiments, includes a series of controls 1620for accessing various screens such as the illustrated screen shot (e.g.,through using a home control 1620 a). The user in some embodiments, mayselect a help control 1620 d to obtain further information regarding thefeatures of the GUI 1600 and the automated instrument. In someimplementations, the user selects a settings control 1620 e to accesssettings options for desired processes and/or the GUI 1600 such as, insome examples, time zone, language, units, network access options. Apower control 1620 f, when selected, allows the user to power down theautomated instrument.

Turning to the protocol pane 1602, in some implementations, a userselects a protocol (e.g., script or work flow) for execution by theautomated instrument by entering the protocol in a protocol entry field1612 (or, alternatively, drop-down menu). In other embodiments, theprotocol may be selected through a separate user interface screen,accessed for example by selecting the script control 1620 b. In anotherexample, the automated instrument may select the protocol and present itin the protocol entry field 1612. For example, the processing system ofthe automated instrument may scan machine-readable indicia positioned onone or more cartridges loaded into the automated instrument to determinethe appropriate protocol. As illustrated, the “Microbe_Kitl (1.0.2)”protocol has been selected, which may correspond to a kit of cartridgesand other disposable supplies purchased for use with the automatedinstrument.

In some implementations, the protocol pane 1602 further includes a startcontrol 1614 a and a stop control 1614 b to control execution of theprotocol presented in the protocol entry field 1612. The GUI 1600 may beprovided on a touch screen interface, for example, where touch selectionof the start control 1614 a starts cell processing, and selection of thestop control 1614 b stops cell processing.

Turning to the run status pane 1604, in some implementations a chart1616 illustrates stages of the processing of the protocol identified inthe protocol pane 1602. For example, a portion of run completion 1618 ais illustrated in blue, while a portion of current stage 1618 b isillustrated in green, and any errors 1618 c are flagged with markersextending from the point in time along the course of the portion of therun completion 1618 a where the error occurred. A message region 1618 dpresents a percentage of run completed, a percentage of stage completed,and a total number of errors. In some embodiments, upon selection of thechart 1616, the user may be presented with greater details regarding therun status such as, in some examples, identification of the type oferror, a name of the current processing stage (e.g., nucleic acidassembly, purification, cell growth, filtration, transformation,recovery, etc.), and a listing of processing stages within the run.Further, in some embodiments a run completion time message indicates adate and time at which the run is estimated to complete. In someembodiments (not shown), the run status pane 1604 additionallyillustrates an estimated time at which user intervention will berequired (e.g., cartridge replacement, solid waste disposal, liquidwaste disposal, etc.).

In some implementations, the run status pane 1604 includes a pausecontrol 1624 for pausing cell processing. The user may select to pausethe current run, for example, to correct for an identified error or toconduct manual intervention such as waste removal.

The temperature pane 1606, in some embodiments, illustrates a series oficons 1126 with corresponding messages 1628 indicating temperaturesettings for various apparatus of the automated instrument. The icons,from left to right, may represent an FTEP module 1626 a (e.g., FTEPdevice associated with the reagent cartridge 1122 of FIG. 11E), apurification module 1626 b, a first growth module 1626 c, a secondgrowth module 1626 d, and a filtration module 1626 e. The correspondingmessages 1628 a-e identify a present temperature, low temperature, andhigh temperature of the corresponding module (e.g., for this stage orthis run). In selecting one of the icons 1626, in some embodiments, agraphic display of temperature of time may be reviewed.

Beneath the temperature pane, in some implementations, a series of panesidentify present status of a number of modules. For example, theelectroporation pane 1608 represents status of a transformation module,while the cell growth pane 1610 represents the status of a growthmodule. In some embodiments, the panes presented here identify status ofa presently operational module (e.g., the module involved in cellprocessing in the current stage) as well as the status of any moduleswhich have already been utilized during the current run (as illustrated,for example, in the run status pane 1604). Past status information, forexample, may present to the user information regarding the parametersused in the prior stage(s) of cell processing.

Turning to the electroporation pane 1608, in some implementations,operational parameters 1630 a of volts, milliamps, and joules arepresented. Additionally, a status message 1632 a may identify additionalinformation regarding the functioning of the transformation module suchas, in some examples, an error status, a time remaining for processing,or contents of the module (e.g., materials added to the module). In someimplementations, an icon 1634 a above the status message 1632 a will bepresented in an active mode (e.g., colorful, “lit up”, in bold, etc.)when the corresponding module is actively processing. Selection of theicon 1634 a, in some embodiments, causes presentation of a graphicdisplay of detailed information regarding the operational parameters1630 a.

Turning to the cell growth pane 1610, in some implementations,operational parameters 1630 b of OD and hours of growth are presented.Additionally, a status message 1632 b may identify additionalinformation regarding the functioning of the growth module such as, insome examples, an error status, a time remaining for processing, orcontents of the module (e.g., materials added to the module). In someimplementations, an icon 1634 b above the status message 1632 b will bepresented in an active mode (e.g., colorful, “lit up”, in bold, etc.)when the corresponding module is actively processing. Selection of theicon 1634 b, in some embodiments, causes presentation of a graphicdisplay of detailed information regarding the operational parameters1630 b.

A hardware description of an example processing system and processingenvironment according to exemplary embodiments is described withreference to FIG. 14. In FIG. 14, the processing system 1410 includes aCPU 1408 which performs a portion of the processes described above. Forexample, the CPU 1408 may manage the processing stages of the method1400 of FIG. 14 and/or the workflows of FIGS. 15A-C. The process dataand, scripts, instructions, and/or user settings may be stored in memory1402. These process data and, scripts, instructions, and/or usersettings may also be stored on a storage medium disk 1404 such as aportable storage medium (e.g., USB drive, optical disk drive, etc.) ormay be stored remotely. For example, the process data and, scripts,instructions, and/or user settings may be stored in a locationaccessible to the processing system 1410 via a network 1428. Further,the claimed advancements are not limited by the form of thecomputer-readable media on which the instructions of the inventiveprocess are stored. For example, the instructions may be stored in FLASHmemory, RAM, ROM, or any other information processing device with whichthe processing system 1410 communicates, such as a server, computer,smart phone, or other hand-held computing device.

Further, components of the claimed advancements may be provided as autility application, background daemon, or component of an operatingsystem, or combination thereof, executing in conjunction with CPU 1408and an operating system such as with other computing systems known tothose skilled in the art.

CPU 1408 may be an ARM processor, system-on-a-chip (SOC),microprocessor, microcontroller, digital signal processor (DSP), or maybe other processor types that would be recognized by one of ordinaryskill in the art. Further, CPU 1408 may be implemented as multipleprocessors cooperatively working in parallel to perform the instructionsof the inventive processes described above.

The processing system 1410 is part of a processing environment 1400. Theprocessing system 1410 in FIG. 14 also includes a network controller1406 for interfacing with the network 1428 to access additional elementswithin the processing environment 1400. As can be appreciated, thenetwork 1428 can be a public network, such as the Internet, or a privatenetwork such as an LAN or WAN network, or any combination thereof andcan also include PSTN or ISDN sub-networks. The network 1428 can bewireless such as a cellular network including EDGE, 3G and 4G wirelesscellular systems. The wireless network can also be Wi-Fi, Bluetooth, orany other wireless form of communication that is known.

The processing system 1410 further includes a general purpose I/Ointerface 1412 interfacing with a user interface (e.g., touch screen)1416, one or more sensors 1414, and one or more peripheral devices 1418.The peripheral I/O devices 1418 may include, in some examples, a videorecording system, an audio recording system, microphone, externalstorage devices, and/or external speaker systems. The one or moresensors 1414 may include one or more of a gyroscope, an accelerometer, agravity sensor, a linear accelerometer, a global positioning system, abar code scanner, a QR code scanner, an RFID scanner, a temperaturemonitor, and a lighting system or lighting element.

The general purpose storage controller 1424 connects the storage mediumdisk 1404 with communication bus 1440, such as a parallel bus or aserial bus such as a Universal Serial Bus (USB), or similar, forinterconnecting all of the components of the processing system. Adescription of the general features and functionality of the storagecontroller 1424, network controller 1406, and general purpose I/Ointerface 1412 is omitted herein for brevity as these features areknown.

The processing system 1410, in some embodiments, includes one or moreonboard and/or peripheral sensors 1414. The sensors 1414, for example,can be incorporated directly into the internal electronics and/or ahousing of the automated multi-module processing instrument. A portionof the sensors 1414 can be in direct physical contact with the I/Ointerface 1412, e.g., via a wire; or in wireless contact e.g., via aBluetooth, Wi-Fi or NFC connection. For example, a wirelesscommunications controller 1426 may enable communications between one ormore wireless sensors 1414 and the I/O interface 1412. Furthermore, oneor more sensors 1414 may be in indirect contact e.g., via intermediaryservers or storage devices that are based in the network 1428; or in(wired, wireless or indirect) contact with a signal accumulatorsomewhere within the automated instrument, which in turn is in (wired orwireless or indirect) contact with the I/O interface 1412.

A group of sensors 1414 communicating with the I/O interface 1412 may beused in combination to gather a given signal type from multiple placesin order to generate a more complete map of signals. One or more sensors1414 communicating with the I/O interface 1412 can be used as acomparator or verification element, for example to filter, cancel, orreject other signals.

In some embodiments, the processing environment 1800 includes acomputing device 1438 communicating with the processing system 1410 viathe wireless communications controller 1426. For example, the wirelesscommunications controller 1426 may enable the exchange of emailmessages, text messages, and/or software application alerts designatedto a smart phone or other personal computing device of a user.

The processing environment 1400, in some implementations, includes arobotic material handling system 1422. The processing system 1410 mayinclude a robotics controller 1420 for issuing control signals toactuate elements of the robotic material handling system, such asmanipulating a position of a gantry, lowering or raising a sipper orpipettor element, and/or actuating pumps and valves to cause liquidtransfer between a sipper/pipettor and various vessels (e.g., chambers,vials, etc.) in the automated instrument. The robotics controller 1420,in some examples, may include a hardware driver, firmware element,and/or one or more algorithms or software packages for interfacing theprocessing system 1410 with the robotics material handling system 1422.

In some implementations, the processing environment 1410 includes one ormore module interfaces 1432, such as, in some examples, one or moresensor interfaces, power control interfaces, valve and pump interfaces,and/or actuator interfaces for activating and controlling processing ofeach module of the automated multi-module processing system. Forexample, the module interfaces 1432 may include an actuator interfacefor the drive motor of rotating cell growth device 1350 (FIGS. 13C and13D) and a sensor interface for the detector board 1372 that sensesoptical density of cell growth within rotating growth vial 1300. Amodule controller 1430, in some embodiments, is configured to interfacewith the module interfaces 1432. The module controller 1430 may includeone or many controllers (e.g., possibly one controller per module,although some modules may share a single controller). The modulecontroller 1430, in some examples, may include a hardware driver,firmware element, and/or one or more algorithms or software packages forinterfacing the processing system 1410 with the module interfaces 1432.

The processing environment 1410, in some implementations, includes athermal management system 1436 for controlling climate conditions withinthe housing of the automated multi-module processing system. The thermalmanagement system 1436 may additional control climate conditions withinone or more modules of the automated instrument. The processing system1410, in some embodiments, includes a temperature controller 1434 forinterfacing with the thermal management system 1436. The temperaturecontroller 1434, in some examples, may include a hardware driver,firmware element, and/or one or more algorithms or software packages forinterfacing the processing system 1410 with the thermal managementsystem 1436.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example 1: Production and Transformation of Electrocompetent E. coli

For testing transformation of the FTEP device, such as the FTEP deviceconfigured as shown in FIGS. 10B-10D (vi), electrocompetent E. colicells were created. To create a starter culture, 6 ml volumes of LBchlor-25 (LB with 25 μg/ml chloramphenicol) were transferred to 14 mlculture tubes. A 25 μl aliquot of E. coli was used to inoculate the LBchlor-25 tubes. Following inoculation, the tubes were placed at a 45°angle in the shaking incubator set to 250 RPM and 30° C. for overnightgrowth, between 12-16 hrs. The OD600 value should be between 2.0 and4.0. A 1:100 inoculum volume of the 250 ml LB chlor-25 tubes weretransferred to four sterile 500 ml baffled shake flasks, i.e., 2.5 mlper 250 ml volume shake flask. The flasks were placed in a shakingincubator set to 250 RPM and 30° C. The growth was monitored bymeasuring OD600 every 1 to 2 hr. When the OD600 of the culture wasbetween 0.5-0.6 (approx. 3-4 hrs), the flasks were removed from theincubator. The cells were centrifuged at 4300 RPM, 10 min, 4° C. Thesupernatant was removed, and 100 ml of ice-cold 10% glycerol wastransferred to each sample. The cells were gently resuspended, and thewash procedure performed three times, each time with the cellsresuspended in 10% glycerol. After the fourth centrifugation, the cellresuspension was transferred to a 50 ml conical Falcon tube andadditional ice-cold 10% glycerol added to bring the volume up to 30 ml.The cells were again centrifuged at 4300 RPM, 10 min, 4° C., thesupernatant removed, and the cell pellet resuspended in 10 ml ice-coldglycerol. The cells are aliquoted in 1:100 dilutions of cell suspensionand ice-cold glycerol.

The comparative electroporation experiment was performed to determinethe efficiency of transformation of the electrocompetent E. coli usingthe embodiment of the FTEP device shown at (ii), (iii), and (vi) ofFIGS. 10B and 10C and (ii) and (vi) of FIG. 10D. The flow rate wascontrolled with a pressure control system. The suspension of cells withDNA was loaded into the FTEP inlet reservoir. The transformed cellsflowed directly from the inlet and inlet channel, through the flowchannel, through the outlet channel, and into the outlet containingrecovery medium. The cells were transferred into a tube containingadditional recovery medium, placed in an incubator shaker at 30° C.shaking at 250 rpm for 3 hours. The cells were plated to determine thecolony forming units (CFUs) that survived electroporation and failed totake up a plasmid and the CFUs that survived electroporation and took upa plasmid. Plates were incubated at 30° C.; E. coli colonies werecounted after 24 hrs.

The flow-through electroporation experiments were benchmarked against 2mm electroporation cuvettes (Bulldog Bio, Portsmouth, N.H.) using an invitro high voltage electroporator (NEPAGENE™ ELEPO21). Stock tubes ofcell suspensions with DNA were prepared and used for side-to-sideexperiments with the NEPAGENE™ and the flow-through electroporation. Theresults are shown in FIG. 19A. In FIG. 19A, the left-most bars hatched/// denote cell input, the bars to the left bars hatched \\\ denote thenumber of cells that survived transformation, and the right bars hatched/// denote the number of cells that were actually transformed. The FTEPdevice showed equivalent transformation of electrocompetent E. colicells at various voltages as compared to the NEPAGENE™ electroporator.As can be seen, the transformation survival rate is at least 90% and insome embodiments is at least 95%, 96%, 97%, 98%, or 99%. The recoveryratio (the fraction of introduced cells which are successfullytransformed and recovered) is in certain embodiments at least 0.001 andpreferably between 0.00001 and 0.01. In FIG. 19A the recovery ratio isapproximately 0.0001.

Additionally, a comparison of the NEPAGENE™ ELEPO21 and the FTEP devicewas made for efficiencies of transformation (uptake), cutting, andediting. In FIG. 19B, triplicate experiments were performed where thebars hatched /// denote the number of cells input for transformation,and the bars hatched \\\ denote the number of cells that weretransformed (uptake), the number of cells where the genome of the cellswas cut by a nuclease transcribed and translated from a vectortransformed into the cells (cutting), and the number of cells whereediting was effected (cutting and repair using a nuclease transcribedand translated from a vector transformed into the cells, and using aguide RNA and a donor DNA sequence both of which were transcribed from avector transformed into the cells). In addition, note that innon-editing cell lines, the number of colonies for both the NEPAGENE™electroporator and the FTEP showed equivalen transformationefficiencies. Moreover, it can be seen that the FTEP showed equivalenttransformation, cutting, and editing efficiencies as the NEPAGENE™electroporator.

Example 2: Production and Transformation of Electrocompetent S.Cerevisiae

For further testing transformation of the FTEP device, such as the FTEPdevice configured as shown in FIGS. 10B-10D (vi), S. Cerevisiae cellswere prepared using the methods as generally set forth in Bergkessel andGuthrie, Methods Enzymol., 529:311-20 (2013). Briefly, YFAP media wasinoculated for overnight growth, with 3 ml inoculate to produce 100 mlof cells. Every 100 ml of culture processed resulted in approximately 1ml of competent cells. Cells were incubated at 30° C. in a shakingincubator until they reached an OD600 of 1.5+/−0.1.

A conditioning buffer was prepared using 100 mM lithium acetate, 10 mMdithiothreitol, and 50 mL of buffer for every 100 mL of cells grown andkept at room temperature. Cells were harvested in 250 ml bottles at 4300rpm for 3 minutes, and the supernatant removed. The cell pellets weresuspended in 100 ml of cold 1 M sorbitol, spun at 4300 rpm for 3 minutesand the supernatant once again removed. The cells were suspended inconditioning buffer, then the suspension transferred into an appropriateflask and shaken at 200 RPM and 30° C. for 30 minutes. The suspensionswere transferred to 50 ml conical vials and spun at 4300 rpm for 3minutes. The supernatant was removed and the pellet resuspended in cold1 M sorbitol. These steps were repeated three times for a total of threewash-spin-decant steps. The pellet was suspended in sorbitol to a finalOD of 150+/−20.

A comparative electroporation experiment was performed to determine theefficiency of transformation of the electrocompetent S. Cerevisiae usingthe FTEP device. The flow rate was controlled with a syringe pump(Harvard apparatus PHD ULTRA™ 4400). The suspension of cells with DNAwas loaded into a 1 mL glass syringe (Hamilton 81320 Syringe, PTFE LuerLock) before mounting on the pump. The output from the functiongenerator was turned on immediately after starting the flow. Theprocessed cells flowed directly into a tube with 1M sorbitol withcarbenicillin. Cells were collected until the same volume electroporatedin the NEPAGENE™ had been processed, at which point the flow and theoutput from the function generator were stopped. After a 3-hour recoveryin an incubator shaker at 30° C. and 250 rpm, cells were plated todetermine the colony forming units (CFUs) that survived electroporationand failed to take up a plasmid and the CFUs that survivedelectroporation and took up a plasmid. Plates were incubated at 30° C.Yeast colonies are counted after 48-76 hrs.

The flow-through electroporation experiments were benchmarked against 2mm electroporation cuvettes (Bulldog Bio, Portsmouth, N.H.) using an invitro high voltage electroporator (NEPAGENE™ ELEPO21). Stock tubes ofcell suspensions with DNA were prepared and used for side-to-sideexperiments with the NEPAGENE™ and the flow-through electroporation. Theresults are shown in FIG. 20. The device showed better transformationand survival of electrocompetent S. Cerevisiae at 2.5 kV voltages ascompared to the NEPAGENE™ method. Input is total number of cells thatwere processed.

Example 3: FTEP Pressure Sensing and Flow Rates

The pressure and sensing was also tested using an FTEP devicesubstantially as shown in FIG. 10B-10D(vi) as part of a cartridge deviceas illustrated in FIG. 11E. An inline flow sensor measurement was usedto indicate when, after the liquid containing the cells and DNA flowedthrough the FTEP chip, where the inlet reservoir was emptied.Approximately 65 μL of liquid was loaded into the input reservoir andthe automated FTEP module was powered on. Looking at the graph at thetop of FIG. 20, it can be seen that after a few short startuptransients, the flow rate shows about ˜3 standard cubic centimeters perminute (SCCM) of flow for almost 8 seconds (8000 ms) until it jumps to24 SCCM. This transition occurs at an end of run trigger, which is anindicator that the liquid containing the cells and DNA has beenprocessed through the FTEP device and that air is not flowing throughthe FTEP device. That trigger may constitute detection of an increaseflow rate or a sudden fluctuation (increase or decrease) in the pressureof the air (such as at a conduit leading from a syringe pump). In onepreferred embodiment, the flow sensor in FIG. 20 detects an increase inair flow indicative of the fluid being completely drained from the inputreservoir. At this point, pressure may be reversed to allow a multi-passelectroporation procedure; that is, cells to electroporated may be“pulled” from the inlet toward the outlet for one pass ofelectroporation, and once the inlet reservoir is emptied, the sensor mayreverse the pressure where the liquid and cells/DNA is “pushed” from theoutlet end of the flow-through FTEP device toward the inlet end to passbetween the electrodes again for another pass of electroporation. Thisprocess may be repeated one to many times. Alternatively, the pressuremay be stopped entirely and the transformed cells in the outletretrieved.

The multi-cycle approach may be particularly advantageous in that itlimits the dwell time of the cells and nucleic acids in the electricfiled which may in turn prevent cell damage and increase survival rates.The back-and-forth process may be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 times. FIG. 20 at bottom shows a simple depiction of the pressuresystem and FTEP. The pressure manifold is mated to theupwardly-extending reservoirs via one or more complementary seals orgaskets disposed on the manifold or the reservoirs.

While certain embodiments have been described, these embodiments havebeen presented by way of example only and are not intended to limit thescope of the present disclosures. Indeed, the novel methods,apparatuses, modules, instruments and systems described herein can beembodied in a variety of other forms; furthermore, various omissions,substitutions and changes in the form of the methods, apparatuses,modules, instruments and systems described herein can be made withoutdeparting from the spirit of the present disclosures. The accompanyingclaims and their equivalents are intended to cover such forms ormodifications as would fall within the scope and spirit of the presentdisclosures.

The invention claimed is:
 1. An automated instrument comprising: ahousing configured to house all of some modules; one or more receptaclesconfigured to receive nucleic acids and cells; a growth moduleconfigured to grow the cells; a cell concentration module configured toconcentrate and render the cells electrocompetent; a flow-throughelectroporation (FTEP) module configured to introduce the nucleic acidsinto the cells; a selection module configured to select for transformedcells; and a processor configured to operate the automated instrumentbased on user input and/or selection of a pre-programmed script; and anautomated liquid handling system to move liquids from the one or morereceptacles to the growth module, from the growth module to the cellconcentration module, from the cell concentration module to the FTEPmodule, and from the FTEP module to the selection module, all withoutuser intervention.
 2. The automated instrument of claim 1, wherein theFTEP module comprises: a. a first inlet and a first inlet channel forintroducing a fluid comprising cells and nucleic acids into the FTEPmodule; b. an outlet and an outlet channel for removing a fluidcomprising transformed cells from the FTEP module; c. a flow channelintersecting and positioned between the first inlet channel and theoutlet channel; and d. two or more electrodes positioned in the flowchannel between the intersection of the flow channel with the firstinlet channel and the intersection of the flow channel with the outletchannel; wherein the electrodes apply one or more electric pulses to thecells in the fluid as they pass through the flow channel therebyintroducing the nucleic acid into the cells in the fluid.
 3. Theautomated instrument of claim 2 wherein the FTEP module furthercomprises a reservoir connected to the first inlet for introducing thecells in fluid into the FTEP module and a reservoir connected to theoutlet for removing transformed cells from the FTEP module.
 4. Theautomated instrument of claim 2 wherein the FTEP module furthercomprises a second inlet and a second inlet channel and furthercomprising a reservoir connected to the second inlet.
 5. The automatedinstrument of claim 4 wherein the second inlet and second inlet channelof the FTEP module are located between the first inlet and first inletchannel and the electrodes of the FTEP module.
 6. The automatedinstrument of claim 4 wherein the second inlet and second inlet channelof the FTEP module are located between the electrodes and the outletchannel and outlet of the FTEP module.
 7. The automated instrument ofclaim 1 wherein the selection module comprises an antibiotic.
 8. Theautomated instrument of claim 1 further comprising a reagent cartridgeand the FTEP module is a component of the reagent cartridge.
 9. Theautomated instrument of claim 2 wherein the FTEP module furthercomprises a filter between the first inlet channel and the electrodes.10. The automated instrument of claim 1, wherein device is configuredfor use with bacterial, yeast and mammalian cells.
 11. The automatedinstrument of claim 1, wherein the cell concentration module comprises afilter.
 12. An automated instrument comprising: a housing configured tohouse all of some modules; one or more receptacles configured to receivenucleic acids and cells; a growth module configured to grow the cells; acell concentration module configured to concentrate and render the cellselectrocompetent; a flow-through electroporation (FTEP) moduleconfigured to introduce the nucleic acids into the cells, wherein theFTEP module comprises a first inlet and a first inlet channel forintroducing a fluid comprising the cells and nucleic acids into the FTEPmodule; an outlet and an outlet channel for removing a fluid comprisingtransformed cells from the FTEP module; a flow channel intersecting andpositioned between the first inlet channel and the outlet channel; andtwo or more electrodes positioned in the flow channel between theintersection of the flow channel with the first inlet channel and theintersection of the flow channel with the outlet channel, wherein theelectrodes apply one or more electric pulses to the cells in the fluidas they pass through the flow channel thereby introducing the nucleicacid into the cells in the fluid, and wherein the FTEP further comprisesa filter between the first inlet channel and the electrodes; a selectionmodule configured to select for transformed cells; and a processorconfigured to operate the automated instrument based on user inputand/or selection of a pre-programmed script; and an automated liquidhandling system to move liquids from the one or more receptacles to thegrowth module, from the growth module to the cell concentration module,from the cell concentration module to the FTEP module, and from the FTEPmodule to the selection module, all without user intervention.
 13. Theautomated instrument of claim 12 wherein the FTEP module furthercomprises a second inlet and a second inlet channel and furthercomprising a reservoir connected to the second inlet for introducing thenucleic acids into the FTEP module.
 14. The automated instrument ofclaim 13 wherein the second inlet and second inlet channel of the FTEPmodule are located between the first inlet and first inlet channel andthe electrodes of the FTEP module.
 15. The automated instrument of claim13 wherein the second inlet and second inlet channel of the FTEP moduleare located between the electrodes and the outlet channel and outlet ofthe FTEP module.
 16. The automated instrument of claim 12 wherein theelectrodes of the FTEP module are from 5 mm to 50 cm in diameter. 17.The automated instrument of claim 12 wherein a narrowest part of theflow channel width of the FTEP module is from 10 μM to 5 mm.
 18. Theautomated instrument of claim 12, further comprising a reagentcartridge.
 19. The automated instrument of claim 18, wherein the FTEPmodule is a component of the reagent cartridge.
 20. The automatedinstrument of claim 19, wherein the selection module comprises anantibiotic.